CHAPTER 1 CHAPTER 17 Fluorophores and Probes for Their ... · Molecular Probes ™ Handbook A Guide to Fluorescent Probes and Labeling Technologies ... BioProbes® Journal of Cell

CHAPTER 17

Probes for Signal Transduction

Molecular Probes™ HandbookA Guide to Fluorescent Probes and Labeling Technologies

11th Edition (2010)

CHAPTER 1

Fluorophores and Their Amine-Reactive Derivatives

The Molecular Probes® HandbookA GUIDE TO FLUORESCENT PROBES AND LABELING TECHNOLOGIES11th Edition (2010)

Molecular Probes® Resources

Molecular Probes® Handbook (online version)Comprehensive guide to �uorescent probes and labeling technologies

lifetechnologies.com/handbook

Fluorescence SpectraViewerIdentify compatible sets of �uorescent dyes and cell structure probes

lifetechnologies.com/spectraviewer

BioProbes® Journal of Cell Biology ApplicationsAward-winning magazine highlighting cell biology products and applications

lifetechnologies.com/bioprobes

Access all Molecular Probes® educational resources at lifetechnologies.com/mpeducate

Molecular Probes ResourcesMolecular Probes Handbook (online version)Comprehensive guide to fl uorescent probes and labeling technologiesthermofi sher.com/handbook

Molecular Probes Fluorescence SpectraViewerIdentify compatible sets of fl uorescent dyes and cell structure probesthermofi sher.com/spectraviewer

BioProbes Journal of Cell Biology ApplicationsAward-winning magazine highlighting cell biology products and applicationsthermofi sher.com/bioprobes

Access all Molecular Probes educational resources at thermofi sher.com/probes

http://thermofisher.com/handbook

http://thermofisher.com/spectraviewer

http://thermofisher.com/bioprobes

http://thermofisher.com/probes

773www.invitrogen.com/probes

The Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling TechnologiesIMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

SEV

ENTE

EN

CHAPTER 17

Probes for Signal Transduction

17.1 Introduction to Signal Transduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 775

17.2 Calcium Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 776

Inositol Triphosphate Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 776

D-myo-1,4,5-Inositol Triphosphate and Caged D-myo-1,4,5-Inositol Triphosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 776

Fluorescent Heparin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 776

Caged Ca2+ and Caged Ca2+ Chelators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 776

NP-EGTA: A Caged Ca2+ Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 776

DMNP-EDTA: A Caged Ca2+ Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 777

Diazo-2: A Photoactivatable Ca2+ Knockdown Reagent. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 777

Other Probes for Calcium Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 777

Thapsigargin and Fluorescent Thapsigargin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 777

Luminescent Calcium Analog . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 777

Data Table 17.2 Calcium Regulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 778

Product List 17.2 Calcium Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 778

17.3 Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins . . . . . . . 779

Protein Kinase Probes and Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 779

Antibody Beacon™ Tyrosine Kinase Assay Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 779

Fluorescent Polymyxin B Analogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 780

Hypericin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 780

Protein Phosphatase Assay Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 781

RediPlate™ 96 EnzChek® Tyrosine Phosphatase Assay Kits. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 781

RediPlate™ 96 EnzChek® Serine/Threonine Phosphatase Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 781

Pro-Q® Diamond Phosphoprotein/Phosphopeptide Microarray Stain Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 782

Adenylate Cyclase Assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783

Adenylate Cyclase Probe: BODIPY® FL Forskolin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783

cAMP Chemiluminescent Immunoassay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783

Nucleotide Analogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783

Alexa Fluor® cAMP and Alexa Fluor® ATP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 784

BODIPY® Ribonucleotide Di- and Triphosphates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 784

Nonhydrolyzable BODIPY® ATP and GTP Analogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 785

N-Methylanthraniloyl (MANT) Nucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 785

Ethenoadenosine Nucleotide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 786

Trinitrophenyl (TNP) Nucleotides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 786

Caged Nucleotides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 787

BzBzATP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 787

Data Table 17.3 Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 788

Product List 17.3 Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 789

The Molecular Probes™ Handbook: A Guide to Fluorescent Probes and Labeling Technologies

IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

thermofi sher.com/probes

Chapter 17 — Probes for Signal Transduction



17.4 Probes for Lipid Metabolism and Signaling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 790

Phospholipase A1 and A2 Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 791

PED-A1 Phospholipase A1 Substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 791

EnzChek® Phospholipase A1 Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 792

Red/Green BODIPY® PC-A2 Ratiometric Phospholipase A2 Substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 792

EnzChek® Phospholipase A2 Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 792

PED6 Phospholipase A2 Substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 793

Other BODIPY® Dye Phospholipase A Substrates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 793

Bis-Pyrenyl Phospholipase A Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794

Singly Labeled Pyrenyl and NBD Phospholipase A2 Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794

ADIFAB Indicator: A Di�erent View of Phospholipase A Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794

Phospholipase C Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 795

EnzChek® Direct Phospholipase C Assay Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 795

Amplex® Red Phosphatidylcholine-Speci�c Phospholipase C Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 795

Bacillus cereus PI-PLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796

Phospholipase D Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796

Amplex® Red Phospholipase D Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796

Fluorescent Substrates for Phospholipase D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797

EnzChek® Lipase Substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797

Anti-Phosphoinositide Monoclonal Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797

Sphingolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797

BODIPY® Sphingolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 798

NBD Sphingolipids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 799

Amplex® Red Sphingomyelinase Assay Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 799

ADIFAB Reagent: A Unique Free Fatty Acid Indicator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 800

Data Table 17.4 Probes for Lipid Metabolism and Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 801

Product List 17.4 Probes for Lipid Metabolism and Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 802


IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.thermofisher.com/probes




Section 17.1 Introduction to Signal Transduction

Cells respond to their environment through a complex and interdependent series of signal transduction pathways that frequently begin at the cell membrane. Many cellular receptors are transmembrane proteins with extracellular domains that selectively bind ligands. In response to ligand binding, the receptor’s cytoplasmic domain may change conformation and transmit the signal across the membrane, or individual receptors may aggregate and interact with other membrane proteins in order to generate a response. Transmembrane signals trigger a cascade of events in the cell, which can include changes in intracel-lular Ca2+ levels, enzymatic activity and gene expression (Figure 17.1.1).

Figure 17.1.1 Neurotransmitter receptors linked to second messengers mediating growth responses in neuronal and nonneuronal cells. Abbreviations: RAC/Gs = Receptors coupled to G-proteins that stimulate adenylate cyclase (AC) activity, leading to cAMP formation and enhanced activity of protein kinase A (PKA). RAC/Gi = Receptors coupled to pertussis toxin (PTX)–sensitive G-proteins that inhibit adenylate cyclase activity. RPLC = Receptors promoting the hydrolysis of phosphatidylinositol 4,5-diphosphate (PIP2) to inositol 1,4,5-triphosphate (IP3), which increases intracellular Ca2+, and diacylglycerol (DAG), which activates protein kinase C (PKC). RION = Receptors indirectly promoting ion �uxes due to coupling to various G-proteins. RLG/ION = Receptors that promote ion �uxes directly because they are structurally linked to ion channels (members of the superfamily of ligand-gated ion channel recep-tors). Stimulation of proliferation is most often associated with activation of G-proteins negatively coupled to adenylate cyclase (Gi), or positively coupled to phospholipase C (Gq) or to pertussis toxin–sensitive pathways (Go, Gi). In contrast, activation of neurotransmitter receptors positively coupled to cAMP usually inhibits cell proliferation and causes changes in cell shape indicative of di�erentiation. Reprinted and modi�ed with permission from J.M. Lauder and Trends Neurosci (1993) 16:233. Learn more about gene speci�c products for signaling pathways at www.invitrogen.com/handbook/pathways.

Ion channel–linkedG-protein–coupled receptors

Phospholipase C–linkedG-protein–coupled receptors

Adenylate cyclase–linkedG-protein–coupled receptors

5-HT3 (Na+, K+)Nicotinic (Na+)GABAA (Cl–)Ionotropic glutamate (Ca2+, Na+)

5-HT1A5-HT4

β-AdrenergicD4 Dopaminergic

A2 AdenosineVIP

Calcium can in�uence cellproliferation, neurite elongation,gene expression and cell viability.

5-HT1A (K+)5-HT1C (Cl–)5-HT2 (Cl–)β-Adrenergic (Ca2+, Na+)α2-Adrenergic (Ca2+, K+)Muscarinic (K+, Ca2+)D2 Dopaminergic (Ca2+, K+)GABAB (K+)

5-HT1A (transfected cells)5-HT1C (transfected cells)5-HT2 (transfected cells)α1-AdrenergicMuscarinicMetabotropic glutamate

Gs

Gi

RAC

PTX

+

Na+

K+

Cl–

Ca2+

–

PTX

IonchannelsGs

Gi

GqACGs GoPLC

5-HT1A5-HT1B5-HT1Dαt-AdrenergicMuscarinicD2 DopaminergicA1 AdenosineOpioidGABAB

RIONRPLC

Gi

ATP

PKA

cAMP+Pi Na+

K+

Cl–

Ca2+

Ligand-gated ion channels

Cell proliferation.

Cell proliferation.

RAC/Gs

RAC/Gi

RPLC

RION

RLG/ION

Gene expression (cAMP response elements), protein phosphorylation,changes in process outgrowth, secretion of growth factors from glia.

Neurite elongation.

RLG/ION

DAG +

PKC Ca2+

IP3PIP2

( )

( )

( )

( ) ( ) ( )

( )

17.1 Introduction to Signal Transduction

We o�er several important reagents for studying signal transduction mechanisms, including Ca2+ regulation and second messenger activities. �is chapter focuses on probes for events occurring down-stream from the receptor–ligand interaction. �ese products comple-ment the probes for receptors and ion channels in Chapter 16, as well as the many ion indicators discussed in Chapter 19, Chapter 20 and Chapter 21. Chapter 18 describes our selection of probes for nitric ox-ide research—including nitric oxide donors, nitric oxide synthase in-hibitors and reagents for nitrite detection—as well as for other reactive oxygen species.



thermofisher.com/probes




Section 17.2 Calcium Regulation

Fluorescent HeparinFluorescein-labeled heparin (H7482) is a useful tool for studying

binding of this mucopolysaccharide in cells and tissues.7,8 In addition to its well-known anticoagulant activity,9 heparin binds to the Ins 1,4,5-P3 receptor and inhibits the biological cascade of events mediated by Ins 1,4,5-P3.10 Heparin also binds to thrombin 11 and Alzheimer’s tau protein,12 as well as to blood vessel–associated proteins such as laminin and �bronectin.13 Fluorescence polarization assays using �uorescein-labeled heparin as a tracer provide quantitative assessments of these binding interactions.14 Fluorescein-labeled heparin has also been used to assess the e�cacy of transdermal delivery of heparin by pulsed cur-rent iontophoresis as a potential alternative to conventional subcutane-ous injections.15

Caged Ca2+ and Caged Ca2+ ChelatorsCaged ions and caged chelators can be used to in�uence the ionic

composition of both solutions and cells, particularly for ions such as Ca2+ that are present at low concentrations. �e properties and uses of caged probes are described in Section 5.3.

NP-EGTA: A Caged Ca2+ ReagentDeveloped by Ellis-Davies and Kaplan, the photolabile chelator o-

nitrophenyl EGTA (NP-EGTA) exhibits a high selectivity for Ca2+, a dramatic 12,500-fold decrease in a�nity for Ca2+ upon UV illumination (its Kd increases from 80 nM to >1 mM) and a high photochemical quantum yield 16,17 (~0.2). Furthermore, with a Kd for Mg2+ of 9 mM, NP-caged EGTA does not perturb physiological levels of Mg2+. We of-fer both the potassium salt (N6802) and the acetoxymethyl (AM) ester (N6803) of NP-EGTA. �e NP-EGTA salt can be complexed with Ca2+ to generate a caged calcium complex that will rapidly deliver Ca2+ upon photolysis (Figure 17.2.2). �e cell-permeant AM ester of NP-EGTA does not bind Ca2+ unless the AM esters are removed. It can poten-tially serve as a photolabile bu�er in cells because, once converted to NP-EGTA by intracellular esterases, it will bind Ca2+ with high a�nity until photolyzed with UV light. NP-EGTA has been used to measure the calcium bu�ering capacity of cells.18

17.2 Calcium RegulationIntracellular Ca2+ levels modulate a multitude of vital cellular pro-

cesses—including gene expression, cell viability, cell proliferation, cell motility, cell shape and volume regulation—thereby playing a key role in regulating cell responses to external signals. �ese dynamic changes in Ca2+ levels are regulated by ligand-gated and G-protein–coupled ion channels in the plasma membrane and by mobilization of Ca2+ from intracellular stores. �e generation of cytosolic Ca2+ spikes and oscilla-tions typically involves the coordinated release and uptake of Ca2+ from these stores, mediated by intracellular Ca2+ channels and their response to several second messengers such as Ca2+ itself, cyclic ADP ribose and inositol triphosphate.1–3

�is section includes several Molecular Probes® reagents for studying Ca2+ regulation in live cells. Fluorescent nucleotides, including analogs of ATP, ADP, AMP, GTP, and GDP, are described in Section 17.3. Our GTP analogs may be particularly useful in the assay of G-protein–coupled receptors. Section 17.4 discusses several selective phosopholipase substrates, as well as labeled ceramide and sphingo-myelin probes.

Inositol Triphosphate PathwayD-myo-1,4,5-Inositol Triphosphate and Caged D-myo-1,4,5-Inositol Triphosphate

We o�er the potassium salt of D-myo-inositol 1,4,5-triphosphate (Ins 1,4,5-P3, I3716) for researchers investigating inositol triphosphate–dependent Ca2+ mobilization and signal transduction mechanisms.1 Cytoplasmic Ins 1,4,5-P3 is a potent intracellular second messenger that induces Ca2+ release from membrane-bound stores in many tissues.

NPE-caged Ins 1,4,5-P3 can be used to generate rapid and precisely controlled release of Ins 1,4,5-P3 in intact cells and is widely employed in studies of Ins 1,4,5-P3–mediated second-messenger pathways.4–6 Our NPE-caged Ins 1,4,5-P3 (I23580) is a mixture of the physiologically in-ert, singly esteri�ed P4 and P5 esters (Figure 17.2.1) and does not contain the somewhat physiologically active P1 ester. NPE-caged Ins 1,4,5-P3 exhibits essentially no biological activity prior to photolytic release of the biologically active Ins 1,4,5-P3.

Figure 17.2.1 D-myo-inositol 1,4,5-triphosphate, P4(5)-(1-(2-nitrophenyl)ethyl) ester, tris(triethylammonium) salt (NPE-caged Ins 1,4,5-P3) (I23580).

Figure 17.2.2 NP-EGTA (N6802) complexed with Ca2+. Upon illumination, this complex is cleaved to yield free Ca2+ and two iminodiacetic acid photoproducts. The a�nity of the pho-toproducts for Ca2+ is ~12,500-fold lower than that of NP-EGTA.

Ca2+

Photocleavage_ _ _ _

OO

NO2

OOC COO

N

COOOOC

N







DMNP-EDTA: A Caged Ca2+ Reagent�e �rst caged Ca2+ reagent described by Ellis-Davies and

Kaplan was 1-(4,5-dimethoxy-2-nitrophenyl) EDTA (DMNP-EDTA, D6814), which they named DM-Nitrophen™ 19,20 (now a trademark of Calbiochem-Novabiochem Corp.). Because its structure better resem-bles that of EDTA than EGTA, we named it as a caged EDTA derivative (Figure 17.2.3). Upon illumination, DMNP-EDTA’s Kd for Ca2+ increas-es from 5 nM to 3 mM. �us, photolysis of DMNP-EDTA complexed with Ca2+ results in a pulse of free Ca2+. Furthermore, DMNP-EDTA has signi�cantly higher a�nity for Mg2+ (Kd = 2.5 µM) than does NP-EGTA 19 (Kd = 9 mM). �e photolysis product’s Kd for Mg2+ is ~3 mM, making DMNP-EDTA an e�ective caged Mg2+ source, in addition to its applications for photolytic Ca2+ release.21,22 Photorelease of Ca2+ has been shown to occur in <180 microseconds, with even faster photore-lease of Mg2+.23 Two reviews by Ellis-Davies discuss the uses and limita-tions of DMNP-EDTA.24,25

Diazo-2: A Photoactivatable Ca2+ Knockdown ReagentIn contrast to NP-EGTA and DMNP-EDTA, diazo-2 (D3034) is a

photoactivatable Ca2+ scavenger. Diazo-2 (Figure 17.2.4), which was in-troduced by Adams, Kao and Tsien,26,27 is a relatively weak chelator (Kd for Ca2+ = 2.2 µM). Following �ash photolysis at ~360 nm, however, cy-tosolic free Ca2+ rapidly binds to the diazo-2 photolysis product, which has a high a�nity for Ca2+ (Kd = 73 nM). Microinjecting a relatively low concentration of �uo-3, �uo-4, or one of the Calcium Green™ or Oregon Green® 488 BAPTA indicators (Section 19.3), along with a known quan-tity of diazo-2, permits measurement of the extent of depletion of cyto-solic Ca2+ following photolysis.27–29 Intracellular loading of NP-EGTA, DMNP-EDTA and diazo-2 is best accomplished by patch pipette infu-sion with the carboxylate salt form of the caged compound added to the internal pipette solution at 1–10 mM. �ese reagents are increasingly being applied in vivo for controlled intervention in calcium-regulated fundamental processes in neurobiology 30 and developmental biology.31

Figure 17.2.3 DMNP-EDTA (D6814) complexed with Ca2+. Upon illumination, this complex is cleaved to yield free Ca2+ and two iminodiacetic acid photoproducts. The a�nity of the photoproducts for Ca2+ is ~600,000-fold lower than that of DMNP-EDTA.

Photocleavage_ _

_

N

CH3O

CH3O

NO2

OOC COO

N COO

COO_

Ca2+

Other Probes for Calcium RegulationThapsigargin and Fluorescent Thapsigargin

�apsigargin is a naturally occurring sesquiterpene lactone isolat-ed from the umbelliferous plant �apsia garganica.32 �is tumor pro-moter releases Ca2+ from intracellular stores by speci�cally inhibiting the sarcoplasmic reticulum Ca2+-ATPase 33,34 (SERCA); it does not di-rectly a�ect plasma membrane Ca2+-ATPases, Ins 1,4,5-P3 production or protein kinase C activity.35,36

�apsigargin is available in 1 mg units (T7458) and specially pack-aged in 20 vials containing 50 µg each (T7459). We have also prepared the green-�uorescent BODIPY® FL thapsigargin (B7487, Figure 17.2.5) and red-�uorescent BODIPY® TR-X thapsigargin (B13800, Figure 17.2.6). BODIPY® FL thapsigargin has proven useful for imaging the intra-cellular localization of thapsigargin during store-operated calcium entry (SOCE) 37 and for imaging SERCA depletion in injured sensory neurons.38

Luminescent Calcium Analog�e trivalent lanthanide terbium (III), which is supplied as its chlo-

ride salt (T1247), is a luminescent analog of Ca2+ that can be used to study structure–function relationships in Ca2+-binding proteins such as calmodulin, oncomodulin, lactalbumin and ATPases.39–41 �e long-lived luminescence of Tb3+ has also been use to probe Ca2+-binding sites of alkaline phosphatase,42 glutamine synthetase,43 integrins,39 protein kinase C 44 and ryanodine-sensitive Ca2+ channels.45 Tb3+ reportedly binds most strongly to the I and II sites of calmodulin.46

Figure 17.2.5 BODIPY® FL thapsigargin (B7487).

Figure 17.2.6 BODIPY® TR-X thapsigargin (B13800).Figure 17.2.4 Diazo-2, tetrapotassium salt (D3034).








DATA TABLE 17.2 CALCIUM REGULATIONCat. No. MW Storage Soluble Abs EC Em Solvent NotesB7487 854.75 FF,D,L DMSO 503 85,000 511 MeOHB13800 1100.04 FF,D,L DMSO 589 62,000 616 MeOHD3034 710.86 F,D,LL pH >6 369 18,000 none pH 7.2 1, 3, 4D6814 473.39 D,LL DMSO 348 4200 none pH 7.2 1, 4, 5H7482 ~18,000 FF,D,L H2O 493 ND 514 pH 8 6, 7I3716 648.64 F,D H2O <250 noneI23580 872.82 FF,D,LL H2O 264 4200 none H2O 1, 2, 8N6802 653.81 FF,D,LL pH >6 260 3500 none pH 7.2 1, 2, 4, 9N6803 789.70 FF,D,LL DMSO 250 4200 none MeCN 10, 11T1247 373.38 D H2O 270 4700 545 H2O 12, 13T7458 650.76 F,D DMSO, EtOH <300 noneT7459 650.76 F,D DMSO, EtOH <300 noneFor de�nitions of the contents of this data table, see “Using The Molecular Probes® Handbook” in the introductory pages.Notes

1. All photoactivatable probes are sensitive to light. They should be protected from illumination except when photolysis is intended.2. This compound has weaker visible absorption at >300 nm but no discernible absorption peaks in this region.3. The Ca2+ dissociation constant of diazo-2 is 2200 nM before photolysis and 73 nM after ultraviolet photolysis. The absorption spectrum of the photolysis product is similar to that of BAPTA. (J Am

Chem Soc (1989) 111:7957)4. Abs and EC values determined in Ca2+-free solution (100 mM KCl, 10 mM EGTA, 10 mM MOPS, pH 7.2).5. Kd (Ca2+) increases from 5 nM to 3 mM after ultraviolet photolysis. Kd values determined in 130 mM KCl, 10 mM HEPES, pH 7.1. (Proc Natl Acad Sci U S A (1988) 85:6571)6. ND = not determined.7. This product is a multiply labeled bioconjugate. The number of labels per conjugate is indicated on the vial.8. Ultraviolet photolysis of I23580 generates I3716.9. Kd (Ca2+) increases from 80 nM to 1 mM after ultraviolet photolysis. Kd values determined in 100 mM KCl, 40 mM HEPES, pH 7.2. (Proc Natl Acad Sci U S A (1994) 91:187)10. This product is intrinsically a liquid or an oil at room temperature.11. N6803 is converted to N6802 via hydrolysis of its acetoxymethyl ester (AM) groups.12. Absorption and luminescence of T1247 are extremely weak unless it is chelated. Data are for dipicolinic acid (DPA) chelate. The luminescence spectrum has secondary peak at 490 nm.13. MW is for the hydrated form of this product.

REFERENCES

PRODUCT LIST 17.2 CALCIUM REGULATIONCat. No. Product Quantity

A7621 8-amino-cyclic adenosine 5’-diphosphate ribose (8-amino-cADP-ribose) 10 µgB7487 BODIPY® FL thapsigargin 100 µgB13800 BODIPY® TR-X thapsigargin 5 µgC7074 cyclic adenosine 5’-diphosphate ribose, 1-(1-(2-nitrophenyl)ethyl) ester (NPE-caged cADP-ribose) *mixed isomers* 50 µgD3034 diazo-2, tetrapotassium salt *cell impermeant* 1 mgD6814 1-(4,5-dimethoxy-2-nitrophenyl)-1,2-diaminoethane-N,N,N’,N’-tetraacetic acid (DMNP-EDTA) *cell impermeant* 5 mgH7482 heparin, �uorescein conjugate 1 mgI3716 D-myo-inositol 1,4,5-triphosphate, hexapotassium salt (Ins 1,4,5-P3) 1 mgI23580 D-myo-inositol 1,4,5-triphosphate, P4(5)-(1-(2-nitrophenyl)ethyl) ester, tris(triethylammonium) salt (NPE-caged Ins 1,4,5-P3) 25 µgN6803 o-nitrophenyl EGTA, AM (NP-EGTA, AM) *cell permeant* *special packaging* 20 x 50 µgN6802 o-nitrophenyl EGTA, tetrapotassium salt (NP-EGTA) *cell impermeant* 1 mgT1247 terbium(III) chloride, hexahydrate 1 gT7458 thapsigargin 1 mgT7459 thapsigargin *special packaging* 20 x 50 µg

1. Biochim Biophys Acta (2009) 1793:933; 2. Cell (2007) 131:1047; 3. Nat Cell Biol (2009) 11:669; 4. Neuron (2007) 54:611; 5. J Physiol (1995) 487:343; 6. Neuron (1995) 15:755; 7. Biochem Biophys Res Commun (2006) 348:850; 8. Chem Biol (2004) 11:487; 9. J Biol Chem (1992) 267:8857; 10. Biochem J (1994) 302:155; 11. J Biol Chem (1998) 273:34730; 12. Biochemistry (2006) 45:6446; 13. J Biol Chem (1995) 270:18558; 14. J Biol Chem (2008) 283:19389; 15. Pharm Res (2006) 23:114; 16. J Biol Chem (1995) 270:23966; 17. Proc Natl Acad Sci U S A (1994) 91:187; 18. Biochem Biophys Res Commun (1998) 250:786; 19. Proc Natl Acad Sci U S A (1988) 85:6571; 20. Science (1988) 241:842; 21. Methods Cell Biol (1994) 40:31; 22. Neuron (1993) 10:21; 23. Biochemistry (1992) 31:8856; 24. Chem Rev (2008) 108:1603; 25. Nat Methods (2007) 4:619; 26. Biochim

Biophys Acta (1990) 1035:378; 27. J Am Chem Soc (1989) 111:7957; 28. Nature (1994) 371:603; 29. Biophys J (1993) 65:2537; 30. Science (2009) 325:207; 31. Dev Growth Di�er (2009) 51:617; 32. Acta Pharm Suec (1978) 15:133; 33. J Biol Chem (1998) 273:12994; 34. J Biol Chem (1995) 270:11731; 35. Proc Natl Acad Sci U S A (1990) 87:2466; 36. J Biol Chem (1989) 264:12266; 37. J Biol Chem (2007) 282:12176; 38. Anesthesiology (2009) 111:393; 39. Biochemistry (1994) 33:12238; 40. J Biol Chem (1992) 267:13340; 41. Photochem Photobiol (1987) 46:1067; 42. J Photochem Photobiol B (1992) 13:289; 43. Biochemistry (1991) 30:3417; 44. J Biol Chem (1988) 263:4223; 45. J Biol Chem (1994) 269:24864; 46. Biochem Biophys Res Commun (1986) 138:1243.






Section 17.3 Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins

17.3 Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins

�e cascade of cellular events in response to an internal signal or environmental stimulus requires a diversity of molecular participants, ranging from ions to enzymes. Signal transduction pathways frequent-ly activate speci�c protein kinases, leading to the phosphorylation of particular cellular proteins and subsequent initiation of a multitude of cellular responses. Binding and hydrolysis of nucleotides plays a ma-jor role in these activities, and our nucleotide analogs and assays for phosphate-producing enzymes are important tools for signal transduc-tion research and high-throughput screening of compounds that a�ect signal transduction.

We o�er a selection of native and modi�ed biomolecules to aid the researcher in dissecting this highly complex branch of the signal trans-duction process. In addition to the probes below, we have developed the PiPer™ and EnzChek® assay kits for quantitation of inorganic phosphate and pyrophosphate that are extremely useful for following hydrolysis of nucleotides by various enzymes and of phosphate esters by protein phosphatases. �ese kits and other kits to measure ATP by chemilumi-nescence and protein phosphatase activity are described in Section 10.3.

Table 17.1 Invitrogen kinase assay platforms.

Assay Principle References

Adapta® Universal Kinase Assay In the absence of an inhibitor, ADP formed by a kinase reaction will displace an Alexa Fluor® 647 dye–labeled ADPtracer from an Eu3+-labeled anti-ADP antibody, resulting in a decrease in the TR-FRET* signal. In the presence of an inhibitor, the amount of ADP formed by the kinase reaction is reduced, and the resulting intact antibody–tracer interaction produces a high TR-FRET signal.

Antibody Beacon™ Tyrosine Kinase Assay Peptide substrate phosphorylation is detected via competitive displacement of an Oregon Green® 488 dye–labeled ligand from a phosphospeci�c antibody.

1

LanthaScreen® Kinase Activity Assays A terbium (Tb3+)– or europium (Eu3+)–labeled phosphospeci�c antibody binds the phosphorylated �uorescein- or Alexa Fluor® 647 dye–labeled peptide substrate, resulting in an increase in the TR-FRET signal.

2, 3

LanthaScreen® Eu Kinase Binding Assay Binding of an Alexa Fluor® 647 tracer to a kinase is detected by addition of a Eu3+-labeled anti–epitope tag antibody. Binding of the tracer and antibody to a kinase results in a high FRET signal, whereas displacement of the tracer by a kinase inhibitor results in a loss of FRET signal.

4

LanthaScreen® Cellular Assays Detection of phosphorylation or other protein modi�cation event is measured on a TR-FRET–compatible plate reader. Little or no TR-FRET is observed with unstimulated or inhibited cells, whereas stimulated cell samples display high TR-FRET.

5, 6

NDP Sensor Protein This �uorescent ADP/ATP biosensor consists of a recombinant bacterial nucleoside diphosphate kinase site-speci�cally labeled with an environment-sensitive coumarin dye.

7

Omnia® Kinase Assay Fluorescence enhancement of N- or C-terminal 8-hydroxyquinoline �uorophore (Sox) upon chelation of Mg2+ is coupled to phosphorylation of a peptide substrate at an adjacent Ser, Thr or Tyr residue.

8, 9

Z´-LYTE® Kinase Assay Phosphorylation-dependent protease susceptibility of a double-labeled peptide substrate is detected using FRET. 10

CellSensor® Cell Lines CellSensor® assays measure pathway-driven activation of transcription factors using GeneBLAzer® β-lactamase reporter technology. Minimal amounts of β-lactamase are expressed in untreated cells or cells treated with a pathway-speci�c inhibitor. Stimulation of the pathway with a ligand or with a constitutively active mutation in a pathway component leads to activation of downstream transcription factor(s), resulting in β-lactamase reporter gene expression. Cells are loaded with a cell-permeable β-lactamase substrate, and β-lactamase reporter activity is measured on a �uorescence plate reader.

11–13

1. Free Radic Biol Med (2009) 47:983; 2. J Biomol Screen (2009) 14:121; 3. Anal Biochem (2006) 356:108; 4. J Biomol Screen (2009) 14:924; 5. J Biomol Screen (2009) 14:121; 6. Anal Biochem (2008) 372:189; 7. Biochemistry (2001) 40:5087; 8. Anal Biochem (2006) 352:198; 9. J Am Chem Soc (2003) 125:14248; 10. Assay Drug Dev Technol (2002) 1:9; 11. Current Chemical Genomics (2009) 3:1; 12. Mol Biosyst (2009) 5:1039; 13. Mol Cancer (2009) 8:117.* TR-FRET = Time-resolved �uorescence resonance energy transfer. For further information on these assay technologies and other kinase biology products and services, visit www.invitrogen.com/handbook/kinase.

Protein Kinase Probes and AssaysProtein kinases are critical players in signal transduction path-

ways. �e �uorometric assay of kinases, however, is not straightforward because ATP-dependent phosphorylation of a �uorescent peptide sub-strate does not directly lead to appreciable changes in the �uorescence of the product.1,2 We provide an extensive range of assays for protein kinases that utilize a variety of strategies to detect phosphorylation of peptide and protein substrates (Table 17.1).

Antibody Beacon™ Tyrosine Kinase Assay Kit�e Antibody Beacon™ Tyrosine Kinase Assay Kit (A35725) provides

a homogeneous solution assay for measuring the activity of tyrosine kinases and the e�ectiveness of potential inhibitors and modulators.3 �e key to this tyrosine kinase assay is a small-molecule tracer ligand labeled with our bright green-�uorescent Oregon Green® 488 dye. When an anti-phosphotyrosine antibody binds this tracer ligand to form the Antibody Beacon™ detection complex, the �uorescence of the Oregon








HN

HN

NH

OHN

ONH3

HN

O

NH

HN

O

HNHOO

NH3

NH

HN

NH

O

O

O

O

NH3

OH

NH3

O

NH3

O

• Compatibility. �e anti-phosphotyrosine antibody provided in the Antibody Beacon™ Tyrosine Kinase Assay Kit is speci�c for phos-photyrosine residues; assay components such as ATP (up to 1 mM) and reducing agents such as dithiothreitol (DTT, up to 1 mM) do not interfere with this assay. �is anti-phosphotyrosine antibody was se-lected from among several clones to produce the greatest �uorescence enhancement by the kinase-phosphorylated product.

• Reliability. �is tyrosine kinase assay has a broad signal window,4 indicated by a Z´ factor of >0.85.

�e Antibody Beacon™ Tyrosine Kinase Assay Kit comes with all the reagents needed to perform this assay, including:

• Oregon Green® 488 dye–labeled tracer ligand• Anti-phosphotyrosine antibody• Concentrated tyrosine kinase reaction bu�er• Two generic tyrosine kinase substrate solutions: a poly(Glu:Tyr)

solution and a poly(Glu:Ala:Tyr) solution• Dithiothreitol (DTT)• Adenosine triphosphate (ATP)• Phosphotyrosine-containing peptide, phospho-pp60 c-src (521–

533), for use as a reference• Detailed protocols

Each kit provides su�cient reagents to perform ~400 assays using a 50 µL assay volume in a �uorescence microplate reader.

Fluorescent Polymyxin B AnalogsPolymyxin B is a cyclic polycationic peptide antibiotic (Figure

17.3.3) that binds to lipopolysaccharides and anionic lipids.5 Polymyxin B is also a selective inhibitor of protein kinase C, with an IC50 of ~35 µM,6–9 as well as a potent inhibitor of calmodulin, with an IC50 of 80 nM in the presence of 500 µM Ca2+.10 Our �uorescent polymyxin B analogs include those of the green-�uorescent BODIPY® FL 11 and Oregon Green® 514 �uorophores (P13235, P13236), as well as the ultra-violet light–excitable dansyl polymyxin 5 (P13238).

HypericinHypericin (H7476, Figure 17.3.4), an anthraquinone derivative isolat-

ed from plants of the genus Hypericum,12,13 is a potent, selective inhibitor of PKC (IC50 = 1.7 µg/mL = 3.4 µM) useful for probing and manipulating PKC in live cells.14 Hypericin has a variety of pharmacological properties, from antibacterial and antineoplastic activities to antiviral activities 15–18 and induction of apoptosis.19 Hypericin is also a potent photosensitizer, with a quantum yield of 0.75 for the generation of singlet oxygen.20

Figure 17.3.2 Real-time detection capability of the Antibody Beacon™ Tyrosine Kinase Assay Kit (A35725). Fluorescence of the Antibody Beacon™ detection complex in tyrosine kinase assay bu�er was monitored over time. After ~15 seconds, an excess of phosphotyrosine-containing peptide was added to the Antibody Beacon™ detection complex and the o�-rate was calculated.

600

200Fluo

resc

ence

400

800

0

Time (seconds)

20 60 80 1400 40

Koff = 0.14 sec-1

Addition of phosphotyrosine-containing peptide

100 120

Figure 17.3.3 Polymyxin B.

Green® 488 dye is e�ciently quenched. In the presence of a phosphoty-rosine-containing peptide, however, this Antibody Beacon™ detection complex is rapidly disrupted, releasing the tracer ligand and relieving its antibody-induced quenching (Figure 17.3.1). Upon its displacement by a phosphotyrosine residue, the Oregon Green® 488 dye–labeled tracer ligand exhibits an approximately 4-fold �uorescence enhancement, en-abling the detection of as little as 50 nM phosphotyrosine-containing peptide with excellent signal-to-background discrimination. Key ben-e�ts of the Antibody Beacon™ Tyrosine Kinase Assay Kit include:

• Real-time measurements. Unlike many other commercially avail-able tyrosine kinase assays, the Antibody Beacon™ Tyrosine Kinase Assay Kit permits real-time monitoring of kinase activity (Figure 17.3.2). Not only is the Antibody Beacon™ detection complex rap-idly dissociated in the presence of phosphotyrosine residues, but the assay components have been designed to be simultaneously combined, reducing any delay in the measurements.

• Simple detection protocol. Tyrosine kinase activity is measured by a simple increase in �uorescence intensity; no special equipment, additional reagents, or extra steps are required. �is assay is readily compatible with any �uorescence microplate reader.

• Use of natural substrates. �e Antibody Beacon™ tyrosine kinase assay utilizes unlabeled peptide or protein substrates, is compatible wth substrates that are pre-phosphorylated at serine or threonine (but not at tyrosine) residues and is applicable to the assay of a wide variety of kinases.

Figure 17.3.1 Reaction scheme for the tyrosine kinase assay used in the Antibody Beacon™ Tyrosine Kinase Assay Kit (A35725). The unlabeled natural substrate (AIYAE) is phosphory-lated by the tyrosine kinase to AIY(P)AE, which displaces the quenched Oregon Green® 488 dye–labeled peptide from the anti-phosphotyrosine antibody, resulting in a large increase in its �uorescence that is proportional to the amount of AIY(P)AE formed in the reaction.

I pY E

ATP

A A

ADP

A I Y A E

Ligand-antibodydetection complex

Phosphorylated substrateand displaced ligand

Substrate

Tyrosine kinase







Protein Phosphatase Assay KitsRediPlate™ 96 EnzChek® Tyrosine Phosphatase Assay Kits

Protein tyrosine phosphatases (PTP) represent a large family of enzymes that play a very important role in intra- and intercellular signaling. PTPs work antagonistically with protein tyrosine kinases to regulate signal transduction pathways in response to a variety of signals, including hormones and mitogens.3 Our RediPlate™ 96 EnzChek® Tyrosine Phosphatase Assay Kit (R22067) provides researchers with a sensitive and convenient method to monitor PTP and screen PTP inhibitors in a variety of research areas.21–24

�e EnzChek® tyrosine phosphatase assay is based on 6,8-di�uoro-4-methylumbellifer-yl phosphate 25 (DiFMUP, D6567, D22065; Section 10.3). Unlike other end-point tyrosine phosphatase assay kits, the EnzChek® tyrosine phosphatase assay is continuous, allowing researchers to easily measure �uorescence at various time points in order to follow the kinetics of the reaction. Furthermore, the assay is not a�ected by free phosphate and is compatible with most nonionic detergents, resulting in minimal sample processing before analysis. Most importantly, each assay well contains inhibitors to help ensure that the assay is selective for tyrosine phosphatases; other phosphatases, including serine/threonine phosphatases, will not hydrolyze DiFMUP under our assay conditions (Figure 17.3.5). Unlike phosphopeptide-based assays, this DiFMUP-based assay can be used to monitor a variety of tyrosine phosphatases, including PTP-1B and CD-45 (Figure 17.3.5). Tyrosine phosphatase inhibitors can be evalu-ated quantitatively in the assay for their e�ect on tyrosine phosphatase activity.

Each RediPlate™ 96 EnzChek® Tyrosine Phosphatase Assay Kit (R22067) includes:

• One RediPlate™ 96 EnzChek® tyrosine phosphatase assay 96-well microplate• Reaction bu�er• Detailed assay protocols

RediPlate™ 96 EnzChek® Serine/Threonine Phosphatase Assay Kit�e majority of protein phosphorylation occurs on serine and threonine residues, with

<0.01–0.05% on tyrosine residues. Serine/threonine phosphatases represent a large family of enzymes that have been implicated in the regulation of metabolism, transcription, translation, di�erentiation, cell cycle, cytoskeletal dynamics, oncogenesis and signal transduction. �e RediPlate™ 96 EnzChek® Serine/�reonine Phosphatase Assay Kit (R33700) provides a fast, sim-ple and direct �uorescence-based assay for detecting serine/threonine phosphatases and their corresponding modulators and inhibitors 25 (Figure 17.3.6).

As with the RediPlate™ 96 EnzChek® Tyrosine Phosphatase Kit, the substrate incorporated in the RediPlate™ 96 EnzChek® Serine/�reonine Phosphatase Assay Kit is DiFMUP. Inhibitors are included in each assay well to help ensure that the assay is selective for serine/threonine phosphatases; under the prescribed assay conditions, other phosphatases, including tyrosine phosphatases, do not signi�cantly react with the substrate (Figure 17.3.7). Furthermore, unlike phosphopeptide-based assays, this DiFMUP-based assay can be used to monitor a variety of ser-ine/threonine phosphatases including PP-1, PP-2A and PP-2B (Figure 17.3.7). Serine/threonine phosphatase inhibitors can be evaluated quantitatively in the assay for their e�ect on serine/

Figure 17.3.4 Hypericin (H7476).

Figure 17.3.6 Schematic diagram of the method used in the RediPlate™ EnzChek® Phosphatase Assay Kits (R22067, R33700).

Measure �uorescence(excitation/emission ~355/455 nm)

Add reaction buffer, then add sample containing PTPase

O

F

F

OPHO

O

OH

O

CH3DiFMUP

(non�uorescent)

O

F

F

O

CH3

HO

DiFMU(�uorescent)

Incubate20–30 minutes

Figure 17.3.5 Speci�city of the RediPlate™ 96 EnzChek® Tyrosine Phosphatase Assay Kit (R22067). The phosphatases listed in the tables were applied to a RediPlate™ 96 EnzChek® tyrosine phosphatase assay microplate. At the indicated time points, the �uorescence was measured in a �uorescence microplate reader using excitation at 355 ± 20 nm and emission at 460 ± 12.5 nm.

Symbol Enzyme (Class) Enzyme Units*

CD-45 (tyrosine phosphatase) 1 U/mL

PTP-1B (tyrosine phosphatase) 1 mU/mL

PTPase (tyrosine phosphatase) 1 U/mL

Acid phosphatase 1 U/mL

Alkaline phosphatase 1 U/mL

PP2A (ser/thr phosphatase)† 1 U/mL

PP1 (ser/thr phosphatase)† 1 U/mL

PP-2B (ser/thr phosphatase)† 500 U/mL

† Serine theonine phosphatase.* Enzyme unit (U) de�nitions are standard de�nitions for each enzyme.

Time (min)

12

10

8

6

4

2

00 5 10 15 20 25 30 35 40 5045

Fluo

resc

ence

Figure 17.3.7 Speci�city of the RediPlate™ 96 EnzChek® Serine/Threonine Phosphatase Assay Kit (R33700) for serine/threonine phosphatases. The phosphatases listed in the tables were applied at the indicated concentrations to a RediPlate™ 96 EnzChek® serine/threonine phosphatase assay micro-plate. Reactions were incubated at 37°C. After 1 hour, �uores-cence was measured in a �uorescence microplate reader us-ing excitation at 355 ± 20 nm and emission at 460 ± 12.5 nm.

Enzyme (U/ml)

120,000

100,000

80,000

60,000

40,000

20,000

00 0.2 0.4 0.6

Fluo

resc

ence

Symbol Enzyme (Class)

PP-2A (Ser/Thr phosphatase)

PP-1 (Ser/Thr phosphatase)

PP-2B (Ser/Thr phosphatase)

Alkaline phosphatase

Acid phosphatase

LAR (tyrosine phosphatase)








threonine phosphatase activity (Figure 17.3.8). Additional advantages of this RediPlate™ assay include compatibility with nonionic detergents and insensitivity to free phosphate, minimizing sample processing before analysis.

Each RediPlate™ 96 EnzChek® Serine/�reonine Phosphatase Assay Kit includes:

• One RediPlate™ 96 EnzChek® serine/threonine phosphatase assay 96-well microplate• Concentrated reaction bu�er• NiCl2• MnCl2• Dithiothreitol• Detailed assay protocols

To ensure the integrity of the predispensed reagents, the 96-well microplate provided in both RediPlate™ Protein Phosphatase Assay Kits is packaged in a resealable foil pouch and consists of twelve removable strips, each with eight wells (Figure 17.3.9). Eleven of the strips (88 wells) are preloaded with the �uorogenic substrate DiFMUP; the remaining strip, marked with black tabs, contains a dilution series of the DiFMU reference standard for generating a standard curve.

Pro-Q® Diamond Phosphoprotein/Phosphopeptide Microarray Stain Kit�e Pro-Q® Diamond Phosphoprotein/Phosphopeptide Microarray Stain Kit (P33706) pro-

vides a method for selectively staining phosphoproteins or phosphopeptides on microarrays with-out the use of antibodies or radioactivity. �is kit permits direct detection of phosphate groups

Figure 17.3.8 Detection of PP-2A inhibition by okadaic acid using the RediPlate™ 96 EnzChek® Serine/Threonine Phosphatase Assay Kit (R33700). Each reaction contained 50 µM DiFMUP, 10 mU/mL PP-2A and the indicated concen-tration (log scale) of okadaic acid in reaction bu�er contain-ing 50 mM Tris-HCl, 0.1 mM CaCl2, 1 mM NiCl2, 125 µg/mL bovine serum albumin (BSA) and 0.05% Tween® 20. Reactions were incubated at 37°C. After 30 minutes, �uorescence was measured in a �uorescence microplate reader using excita-tion at 355 ± 20 nm and emission at 460 ± 12.5 nm.

Figure 17.3.9 A RediPlate™ 96 microplate.

NOTE 17.1

G-Proteins and GTP Analogs for Binding StudiesWe prepare a wide variety of nucleotide analogs for protein-binding studies; their chemical and

spectral properties are described in Section 17.3. These include various �uorescent, photoa�nity and caged versions of adenosine and guanosine triphosphates, diphosphates and cyclic monophos-phates. The GTP analogs are among the most important probes for the study of G-proteins and G protein–coupled receptors (GPCR). Heterotrimeric guanine nucleotide–binding regulatory proteins transmit a variety of receptor signals to modulate diverse cellular responses,1,2 including apoptosis.3

G-proteins are composed of α-, β- and γ-subunits. Upon receptor stimulus, the α-subunit of the heterotrimeric G-proteins exchanges GDP for GTP and dissociates from the β-γ-subunit complex. The GTP-bound G protein will interact with various second-messenger systems, either inhibiting (Gi) or stimulating (Gs) their activity. Stimulatory G-proteins are permanently activated by cholera toxin, inhibitory G-proteins by pertussis toxin. The α-subunit has a slow intrinsic rate of GTP hydrolysis, and once the GTP is hydrolyzed it reassociates with the β-γ-subunit complex. The GTP hydrolysis by G-proteins is regulated by interactions with GTPase-activating proteins, or GAPs. There is a large family of GAPs for G-proteins known as regulators of G protein signaling or, RGS proteins.4 G-proteins are turned o� when the α-subunit hydrolyzes the GTP, either spon-taneously or upon interaction with a GTPase-activating protein, permitting the heterotrimeric α-β-γ-complex to reassociate.

The GAPs are a diverse group of monomeric GTPases, including ARF, Ran, Ras, Rab, Rac, Rho and Sar, which play an important part in regulating many intracellular processes, such as cytoskeletal organization and secretion. There is less diversity among the β- and γ-subunits, but they may have direct activating e�ects in their own right. Most β- and γ-subunits are posttranslationally modi�ed by myristoylation or isoprenylation, which may alter their association with membranes.

Our �uorescent GTP analogs include:

• Blue-�uorescent MANT-GTP (M12415) and nonhydrolyzable MANT-GMPPNP (M22353)• Green-�uorescent BODIPY® FL guanosine 5’-triphosphate (BODIPY® FL GTP, G12411)• Red-�uorescent BODIPY® TR guanosine 5’-triphosphate (BODIPY® TR GTP, G22351)• Nonhydrolyzable green-�uorescent BODIPY® FL GTP-γ-S thioester (G22183)

1. Annu Rev Biochem (2008) 77:1; 2. Physiol Rev (1999) 79:1373; 3. J Biol Chem (2000) 275:20726; 4. J Biol Chem (1998) 273:1269.







attached to tyrosine, serine or threonine residues in a microarray environment and has been optimized for microarrays with acrylamide gel surfaces. Each Pro-Q® Diamond Phosphoprotein/Phosphopeptide Microarray Stain Kit provides:

• Pro-Q® Diamond phosphoprotein/phosphopeptide microarray stain• Pro-Q® Diamond microarray destain solution• Microarray staining gasket with seal tabs, 10 chambers• Slide holder tube, 20 tubes• Detailed protocols

�e Pro-Q® Diamond Phosphoprotein/Phosphopeptide Microarray Stain Kit is ideal for identifying kinase targets in signal transduction pathways and for phosphoproteomics studies.26

Adenylate Cyclase Assays3 ,́5´-Cyclic AMP (cAMP) is an important second messenger in many

signal transduction pathways, linking activation of cell-surface mem-brane receptors to intracellular responses, and, ultimately, to changes in gene expression. cAMP is synthesized by plasma membrane–bound adenylate cyclase, which is coupled to transmembrane receptors for hor-mones, neurotransmitters and other signaling molecules by heterotri-meric G-proteins. Upon ligand binding, the intracellular receptor do-main of a G-protein–coupled receptor (GPCR) interacts with a G-protein, which then dissociates and activates adenylate cyclase, resulting in an increase in the concentration of intracellular cAMP. Subsequently, cAMP activates cAMP-dependent protein kinases (protein kinase A), which phosphorylate speci�c substrate proteins, including enzymes, structural proteins, transcription factors and ion channels.

Adenylate Cyclase Probe: BODIPY® FL ForskolinForskolin, isolated from Coleus forskohlii, is a potent activator of

adenylate cyclase, the enzyme that catalyzes the formation of cAMP from ATP. Green-�uorescent BODIPY® FL forskolin (B7469, Figure 17.3.10) has been used to visualize adenylyl cyclase internalization and subcellular distribution,27 as well as for the pharmacological character-ization of adenylyl cyclase catalytic subunits.28

cAMP Chemiluminescent Immunoassay Kit�e cAMP Chemiluminescent Immunoassay Kit enables ultrasen-

sitive determination of 3 ,́5´-cyclic AMP (cAMP) levels in cell lysates, providing the highest sensitivity of any commercially available cAMP assay. As few as 60 femtomoles of cAMP can be detected. Furthermore, this assay has a wide dynamic range, detecting from 0.06 to 6000 pi-comoles without the need for sample dilution or manipulations such as acetylation. �is extensive dynamic range is especially important in cell-based assays designed to measure Gs- or Gi-coupled agonist stimulation or inhibition. Intra-assay precision for duplicate samples is typically 5% or less.

�is competitive immunoassay is formatted with maximum �ex-ibility to permit either manual assay or automated high-throughput screening. �e cAMP immunoassay is based on the highly sensitive CSPD® alkaline phosphate substrate, a chemiluminescent 1,2-diox-etane, with Sapphire-II™ luminescence enhancer. �e ready-to-use Figure 17.3.10 BODIPY® FL forskolin (B7469).

substrate ⁄enhancer reagent generates sustained glow light emission that is measured 30 minutes a�er addition. Once the substrate ⁄enhancer reaches the glow signal, the plate can be read for hours with little or no degradation of the signal, facilitating screening protocols in which several plates are compared to each other. In addition, the assay exhibits exceptionally low cross-reactivity with other adenosine-containing or cyclic nucleotides.

�e cAMP Chemiluminescent Immunoassay Kit (2-plate size, C10557; 10-plate size, C10558) is designed for the rapid and sensitive quantitation of cAMP in extracts prepared from mammalian cells cultured in microwell plates. Each kit provides all required reagents, including:

• Alkaline phosphate conjugate of cAMP• Anti-cAMP antibody• cAMP standard• CSPD® substrate and Sapphire™-II luminescence enhancer• Assay and lysis bu�er• Conjugate dilution bu�er• Wash bu�er• Precoated microplates• Detailed protocols

�e cAMP Chemiluminescent Immunoassay Kit is designed for quantitating cellular cAMP for functional assays of receptor activa-tion. It has been used with established cell lines for functional mea-surements with endogenous receptors,29–32 with cell lines containing exogenously expressed ligand receptors,33,34 with primary cells 35,36 and with tissues.37 It has also been used for receptor characterization,38 orphan receptor ligand identi�cation 39 and the characterization of novel chimeric receptors.40 In addition, this assay can be used for high-throughput screening assays 41 of compounds that stimulate or interfere with these signal transduction pathways.

Nucleotide AnalogsNucleotide analogs that serve as substrates or inhibitors of en-

zymes, as well as nucleotide derivatives that selectively bind to regula-tory sites of nucleotide-binding proteins, have been used as structural and mechanistic probes for isolated proteins, reconstituted membrane-bound enzymes, organelles such as mitochondria, and tissues such as skinned muscle �bers.42 More recently, however, these analogs have also been employed to study the e�ects of nucleotides on signal transduction and to screen for compounds that may a�ect signal transduction, such as G protein inhibitors and activators (G-Proteins and GTP Analogs for Binding Studies—Note 17.1).








Figure 17.3.12 Adenosine 5’-triphosphate, BODIPY® FL 2’-(or-3’)-O-(N-(2-aminoethyl)urethane), trisodium salt (BODIPY® FL ATP) (A12410).

We prepare a variety of nucleotide analogs, including:

• Alexa Fluor® derivatives of cAMP for use as probes of type I cAMP-dependent protein ki-nases (PKA I) and Alexa Fluor® 647 ATP (A22362)

• BODIPY® dye–labeled nucleotides for use as enzyme substrates and as long-wavelength probes of nucleotide-binding sites

• Environment-sensitive, blue-�uorescent N-methylanthraniloyl (MANT) nucleotides• Blue-�uorescent ethenoadenosine triphosphate (ε-ATP, E23691)• Environment-sensitive trinitrophenyl (TNP) nucleotides• Caged nucleotides, which are important probes for studying the kinetics and mechanism of

nucleotide-binding proteins because they allow spatial and temporal control of the release of active nucleotide

• Photoa�nity nucleotides for site-selective covalent labeling• Fluorescent ChromaTide® nucleotides and aha-dUTP nucleotides, which are primarily used

for biosynthetic incorporation into DNA or RNA (Section 8.2)

Alexa Fluor® cAMP and Alexa Fluor® ATPOur Alexa Fluor® cAMP analogs are 8-(6-aminohexyl)amino derivatives; similar analogs

have been shown to exhibit a marked preference for binding to type I cAMP-dependent protein kinases (PKA I). We o�er the green-�uorescent Alexa Fluor® 488 cAMP (A35775, Figure 17.3.11) and far-red–�uorescent Alexa Fluor® 647 cAMP (A35777). Alexa Fluor® 488 cAMP was loaded into cells by electroporation and then used to measure intercellular di�usion of cAMP from regulatory to responder T cells via gap junctions.43

�e Alexa Fluor® 647 conjugate of ATP (A22362) comprises the long-wavelength Alexa Fluor® 647 �uorophore linked to the ribose of ATP by a urethane bridge. Validated applica-tions of this probe include �uorescence resonance energy transfer (FRET) analysis (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2) of nucleotide assocation with Na+/K+-ATPase 44 and measurements of the catalytic activity of heavy meromyosin.45

BODIPY® Ribonucleotide Di- and TriphosphatesOur selection of BODIPY® dye–modi�ed ribonucleotides includes:

• BODIPY® FL adenosine 5´-triphosphate (BODIPY® FL ATP, A12410)• BODIPY® TR adenosine 5´-triphosphate (BODIPY® TR ATP, A22352)• BODIPY® TR adenosine 5´-diphosphate (BODIPY® TR ADP, A22359)• BODIPY® FL guanosine 5´-triphosphate (BODIPY® FL GTP, G12411)• BODIPY® TR guanosine 5´-triphosphate (BODIPY® TR GTP, G22351)• BODIPY® FL guanosine 5´-diphosphate (BODIPY® FL GDP, G22360)

Figure 17.3.14 Adenosine 5’-O-(3-thiotriphosphate), BODIPY® FL thioester, sodium salt (BODIPY® FL ATP-γ-S, thioester) (A22184).

Figure 17.3.13 Fluorescence emission spectra of (1) free BODIPY® FL dye in phosphate-bu�ered saline, pH 7.2; (2) BODIPY® FL ATP (A12410); and (3) BODIPY® FL GTP (G12411). Samples were prepared with equal absorbance at the excitation wavelength (488 nm). The areas under the curves are therefore proportional to the relative �uores-cence quantum yields, clearly showing the quenching e�ect caused by interaction of the BODIPY® FL �uorophore with the guanine base of GTP.

Wavelength (nm)550 600500

Fluo

resc

ence

em

issi

on

Ex = 488 nm1

2

3

Figure 17.3.11 Alexa Fluor® 488 8-(6-aminohexyl)aminoadenosine 3’,5’-cyclicmonophosphate, bis(triethylammonium) salt (Alexa Fluor® 488 cAMP) (A35775).

2 (CH3CH2)3NH

CO

NH(CH2)6NHN

NN

N

NH2

O

O OH

OCH2

POO

H2N O NH2

CO

OH

SO3SO3

6

5







�ese mixed-isomer analogs comprise a BODIPY® �uorophore at-tached to the 2´ or 3´ position of the ribose ring via an aminoethylcar-bamoyl linker (Figure 17.3.12). Interactions between the �uorophore and the purine base are evident from the spectroscopic properties of these nucleotide analogs. �e �uorescence quantum yield of BODIPY® FL GTP and BODIPY® FL ATP is signi�cantly quenched in solution (Figure 17.3.13) and increases upon binding to at least some GTP-binding pro-teins.46,47 Similar nucleotide analogs incorporating �uorophores such as �uorescein, tetramethylrhodamine and Cy®3 dye have been primar-ily used for biophysical studies of nucleotide-binding proteins.48 �e BODIPY® dye–labeled nucleotides may be particularly useful for �uo-rescence polarization–based assays of ATP- or GTP-binding proteins.

Nonhydrolyzable BODIPY® ATP and GTP AnalogsAmong the most useful �uorescent nucleotides for protein-binding

studies are those that stoichiometrically bind to ATP- or GTP-binding sites but are not metabolized. We o�er the following nonhydrolyzable BODIPY® nucleotides:

• BODIPY® FL AMPPNP 49 (B22356)• BODIPY® FL ATP-γ-S (A22184, Figure 17.3.14)• BODIPY® FL GTP-γ-S46 (G22183)• BODIPY® 515/530 GTP-γ-S (G35779)• BODIPY® TR GTP-γ-S (G35780)• BODIPY® FL GTP-γ-NH amide (G35778)

�e �uorescence of the BODIPY® GTP-γ-S thioesters is quenched ~90% relative to that of the free dye but is recovered upon protein bind-ing to G-proteins.46 �e green-�uorescent BODIPY® FL GTP-γ-S has been used to detect GTP-binding proteins separated by capillary elec-trophoresis.50 As compared with BODIPY® FL GTP-γ-S thioester, the green-�uorescent BODIPY® 515/530 GTP-γ-S thioester has a greater �uorescence increase upon protein binding. �e BODIPY® TR GTP-γ-S thioester is a red-�uorescent analog with spectral properties similar to the Texas Red® dye.

Although BODIPY® FL GTP-γ-NH amide exhibits less �uorescence enhancement upon protein binding, it is reportedly the best of the three green-�uorescent GTP-γ analogs for directly monitoring nucleotide ex-change.51 �e di�erent linker lengths of the green-�uorescent GTP-γ analogs (six-carbon for BODIPY® FL GTP-γ-NH amide, four-carbon for BODIPY® FL GTP-γ-S and one-carbon for BODIPY® 515/530 GTP-γ-S) may be useful for understanding protein active-site geometries.

In addition to their potential use for binding studies, BODIPY® FL ATP-γ-S and BODIPY® FL GTP-γ-S thioesters are important substrates for Fhit (Figure 17.3.15), a member of the histidine triad superfamily of nucleotide-binding proteins that bind and cleave diadenosine poly-phosphates.52–54 Fhit, one of the most frequently inactivated proteins in lung cancer, functions as a tumor suppressor by inducing apopto-sis.53,55,56 �ese BODIPY® nucleotides should be especially useful for screening potential Fhit inhibitors and activators.

N-Methylanthraniloyl (MANT) Nucleotides�e blue-�uorescent MANT nucleotide analogs of ATP (Figure

17.3.16), AMPPNP, GTP, GMPPNP, ADP and GDP are modi�ed on the ribose moiety, making these probes particularly useful for studying nucleotide-binding proteins that are sensitive to modi�cations of the

Figure 17.3.16 2’-(or-3’)-O-(N-methylanthraniloyl)adenosine 5’-triphosphate, trisodium salt (MANT-ATP) (M12417).

Figure 17.3.15 Principle of �uorescence-based detection of the diadenosine triphosphate hydrolase activity of Fhit using BODIPY® FL GTP-γ-S thioester (G22183) as a substrate analog.

NB

N

FF

H3C

H3C CH2NH C

O

CH2 S

OH

H2N

O

O

P O P O

O

P

OH

OOCH2

O

HN

N

N

NO

OO

OH

H2N

HO P

OH

OOCH2

O

HN

N

N

NO

O

Fhit

BODIPY® FL GTP- -S

BODIPY® FL thiodiphosphate

GMP

+

e

Electron-transfer quenching

NB

N

FF

H3C

H3C CH2NH C

O

CH2 S

O

O

P O P OH

O

O

Weak �uorescence

Strong �uorescence








purine base.57,58 �e compact nature of the MANT �uorophore and its attachment position on the ribose ring results in nucleotide analogs that induce minimal perturbation of nucleotide–protein interactions, as con�rmed by X-ray crystal structures of MANT nucleotides bound to myosin 59 and H–ras p21.60 Furthermore, because MANT �uorescence is sensitive to the envi-ronment of the �uorophore, nucleotide–protein interactions may be directly detectable. �ese properties (Table 17.2) make MANT nucleotides valuable probes of the structure and enzymatic activity of nucleotide-binding proteins.48

Applications for MANT-ATP (M12417), MANT-ADP (M12416) and MANT-AMPPNP (M22354) include analysis of:

• ATPase kinetics of kinesin 61–63 and other microtubule motor-proteins 64,65 using stopped-�ow �uorescence measurements

• Conformation of the myosin subfragment-1 nucleotide-binding site, as indicated by �uores-cence quencher accessibility 66,67

• Interaction of P-glycoprotein ATP-binding sites with drug e�ux–modulating steroids 68

• Myosin ATPase activity in rabbit skeletal muscle 69

• Structural characteristics of the nucleotide-binding site of Escherichia coli DnaB helicase 70,71

Applications for MANT-GTP (M12415), MANT-GDP (M12414) and MANT-GMPPNP (M22353) include analysis of:

• Activation of protein kinases by Rho subfamily GTP-binding proteins 72

• Conformational changes during activation of heterotrimeric G-proteins 73

• E�ects of nucleotide structural modi�cations on binding to H–ras p21 74

• Nucleotide hydrolysis and dissociation kinetics of H–ras p21 and other low molecular weight GTP-binding proteins 57,75–78

• GTP-binding proteins Rab5 and Rab7,79 Raf-1,80 Rho 81,82 and Rac,83,84 as well as Ras-related proteins 57,75

Ethenoadenosine Nucleotide�e ethenoadenosine nucleotides—developed in 1972 by Leonard and collaborators 85,86—

bind like endogenous nucleotides to several proteins. �e properties and applications of ethe-noadenosine and MANT nucleotides have been comprehensively reviewed.48 �e etheno ATP analog (ε-ATP, E23691; Figure 17.3.17) can o�en mimic ATP in both binding and function. �is probe has been used to replace ATP in actin polymerization reactions 87 and is frequently in-corporated in place of the tightly bound actin nucleotide.88,89 It also supports contraction of actomyosin, facilitates the measurement of nucleotide-exchange kinetics in actin 90 and serves as a substrate for myosin, which converts it to ε-ADP.91 Sensitized luminescence of Tb3+ (T1247, Section 14.3) coordinated to ε-ATP is a sensitive probe of binding to the catalytic site of protein disul�de isomerase.92

Trinitrophenyl (TNP) NucleotidesUnlike the etheno derivatives, the free trinitrophenyl (TNP) nucleotides are essentially non-

�uorescent in water. �e TNP nucleotides undergo an equilibrium transition to a semiquinoid structure that has relatively long-wavelength spectral properties; 93–95 this form is only �uores-cent when bound to the nucleotide-binding site of some proteins. �e TNP derivative of ATP frequently exhibits a spectral shi� and �uorescence enhancement upon protein binding and actually binds with higher a�nity than ATP to some proteins. �e broad, long-wavelength ab-sorption of TNP nucleotides makes them useful for FRET studies 96–98 (Fluorescence Resonance

Figure 17.3.18 2’-(or-3’)-O-(trinitrophenyl)adenosine 5’-tri-phosphate, trisodium salt (TNP-ATP) (T7602).

Figure 17.3.17 1,N 6-ethenoadenosine 5’-triphosphate (ε-ATP) (E23691).

Table 17.2 Spectroscopic properties of MANT-nucleotides in aqueous solution (pH 8).

Parameter Value Notes

Absorption maximum 356 nm Stronger absorption at shorter wavelengths (λmax = 255 nm)

Molar extinction coe�cient (ECmax) 5800 cm–1M–1 23,000 cm–1M–1 at 255 nm

Fluorescence emission maximum 448 nm Shifts 10–20 nm shorter in nonpolar solvents and upon binding to most proteins

Fluorescence quantum yield 0.22 Increases in nonpolar solvents and upon binding to most proteins







Energy Transfer (FRET)—Note 1.2). �e TNP derivatives of ATP (TNP-ATP, T7602; Figure 17.3.18), ADP (TNP-ADP, T7601) and AMP (TNP-AMP, T7624) have been used as structural probes for a wide variety of nucleotide-binding proteins.99–101 We have found that chromato-graphically puri�ed TNP nucleotides are unstable during lyophiliza-tion. Consequently, these derivatives are sold in aqueous solution and should be frozen immediately upon arrival.

Caged NucleotidesCaged nucleotides are nucleotide analogs in which the terminal

phosphate is esteri�ed with a blocking group, rendering the molecule biologically inactive. Photolytic removal of the caging group by UV il-lumination results in a pulse of the nucleotide—o�en on a microsecond to millisecond time scale—at the site of illumination. Because photoly-sis ("uncaging") can be temporally controlled and con�ned to the area of illumination, the popularity of this technique is growing. We are supporting this development by synthesizing a variety of caged nucleo-tides, neurotransmitters and Ca2+ chelators. Our current selection of caged nucleotides includes:

• NPE-caged ATP (A1048)• DMNPE-caged ATP (A1049)• NPE-caged ADP (A7056)• DMNB-caged c-AMP (D1037)

Section 5.3 discusses our selection of caged probes and the proper-ties of the di�erent caging groups that we use (Table 5.2).

Researchers investigating the cytoskeleton have bene�ted greatly from advances in caging technology, primarily originating from the work of Trentham, Kaplan and their colleagues.102 NPE-caged ADP (A7056) is a useful probe for studying the e�ect of photolytic release of ADP in muscle �bers 103,104 and isolated sarcoplasmic reticulum.105 Although it is

sometimes di�cult to properly abstract papers that describe experiments with caged ATP because they could be referring to either NPE-caged ATP (A1048), DMNPE-caged ATP (A1049) or earlier caged versions of this nucleotide, most researchers have used NPE-caged ATP.

Because the caged nucleotides may be added to an experimental system at relatively high concentrations, use of the enzyme apyrase was recommended by Sleep and Burton 106 to eliminate any traces of ATP that may be present in the caged ATP probes.107–110 Once the caged ATP solutions have been preincubated with apyrase, the enzyme can be re-moved by centrifugal �ltration.107,109

�ese caged nucleotides are generally cell impermeant and must be microinjected into cells or loaded by other techniques (Table 14.1). Permeabilization of cells with staphylococcal α-toxin or the saponin ester β-escin is reported to make the membrane of smooth muscle cells permeable to low molecular weight (<1000 daltons) mol-ecules, while retaining high molecular weight compounds.111 α-Toxin permeabilization has permitted the introduction of caged nucleotides, including caged ATP (A1048) and caged GTP-γ-S, as well as of caged inositol 1,4,5-triphosphate (NPE-caged Ins 1,4,5; I23580; Section 17.2) into smooth muscle cells.112 Caged inositol 1,4,5-triphosphate has also been successfully loaded in ECV304 cells using electroporation.113

BzBzATPFunctional ion channels can be assembled from both homomeric

and heteromeric combinations of the seven P2X receptor subunits so far identi�ed (P2X1–7). Due to the lack of speci�c agonists or antagonists for P2X receptors, it is di�cult to determine which receptor subtypes mediate particular cellular responses. We o�er one of the most potent and widely used P2X receptor agonists, BzBzATP 114,115 (2´-(or 3´-)O-(4-benzoylbenzoyl)adenosine 5´-triphosphate, B22358). BzBzATP has more general applications for site-directed irreversible modi�cation of nucleotide-binding proteins via photoa�nity labeling.116,117

REFERENCES1. Biochim Biophys Acta (2008) 1784:94; 2. J Am Chem Soc (2009) 131:13286; 3. Free Radic Biol Med (2009) 47:983; 4. J Biomol Screen (1999) 4:67; 5. J Med Chem (2010) 53:1898; 6. Am J Physiol (1989) 256:C886; 7. J Neurochem (1986) 47:1405; 8. Nature (1986) 321:698; 9. J Neurosci (1985) 5:2672; 10. Eur J Pharmacol (1991) 207:17; 11. Antimicrob Agents Chemother (2009) 53:3501; 12. J Pharm Pharmacol (2001) 53:583; 13. Med Res Rev (1995) 15:111; 14. Photochem Photobiol (2006) 82:720; 15. Anticancer Res (1998) 18:4651; 16. Photochem Photobiol (1998) 68:593; 17. Proc Natl Acad Sci U S A (1989) 86:5963; 18. Pharmacol �er (1994) 63:1; 19. J Biol Chem (2002) 277:37718; 20. Photochem Photobiol (1994) 59:529; 21. J Biol Chem (2008) 283:3401; 22. Nat Med (2006) 12:549; 23. Nature (2005) 437:911; 24. Nat Cell Biol (2005) 7:78; 25. Comb Chem High �roughput Screen (2003) 6:341; 26. Proteomics (2003) 3:1244; 27. PLoS Biol (2009) 7:e1000172; 28. J Pharmacol Exp �er (2008) 325:27; 29. J Biol Chem (2005) 280:4048; 30. J Med Chem (2008) 51:1831; 31. Mol Pharmacol (2008) 73:1371; 32. J Biomol Screen (2000) 5:239; 33. J Biochem (2006) 139:543; 34. Mol Endocrinol (2007) 21:700; 35. Kidney Int (2007) 71:738; 36. J Immunol (2005) 174:1073; 37. J Clin Invest (2003) 112:398; 38. J Biol Chem (2004) 279:19790; 39. Nature (2004) 429:188; 40. Proc Natl Acad Sci U S A (2004) 101:1508; 41. Nucleic Acids Res (2003) 31:e130; 42. J Cell Sci (2001) 114:459; 43. J Exp Med (2007) 204:1303; 44. Biochim Biophys Acta (2009) 1794:1549; 45. Biochemistry (2007) 46:7233; 46. J Biol Chem (2001) 276:29275; 47. Anal Biochem (2001) 291:109; 48. Methods Enzymol (1997) 278:363; 49. J Biol Chem (2004) 279:28402; 50. Anal Chem (2003) 75:4297; 51. Proc Natl Acad Sci U S A (2004) 101:2800; 52. Proc Natl Acad Sci U S A (2003) 100:1592; 53. Curr Biol (2000) 10:907; 54. J Biol Chem (2000) 275:4555; 55. Am J Pathol (2000) 156:419; 56. J Natl Cancer Inst (2000) 92:338; 57. Proc Natl Acad Sci U S A (1990) 87:3562; 58. Biochim Biophys Acta (1983)

742:496; 59. J Mol Biol (1997) 274:394; 60. J Mol Biol (1995) 253:132; 61. Biochemistry (1998) 37:792; 62. J Biol Chem (1997) 272:717; 63. Biochemistry (1995) 34:13233; 64. Biochemistry (1996) 35:2365; 65. Biochemistry (1995) 34:13259; 66. Biophys J (1995) 68:142S; 67. Biochemistry (1990) 29:3309; 68. Biochemistry (1997) 36:15208; 69. J Gen Physiol (1995) 106:957; 70. Biophys J (1996) 71:2075; 71. J Cell Biol (1997) 139:63; 72. Biochemistry (1997) 36:1173; 73. J Biol Chem (1994) 269:13771; 74. Biochemistry (1995) 34:593; 75. Biochemistry (1997) 36:4535; 76. Biochemistry (1995) 34:12543; 77. Biochemistry (1995) 34:639; 78. Biochemistry (1993) 32:7451; 79. J Biol Chem (1996) 271:20470; 80. J Biol Chem (2000) 275:22172; 81. Biochemistry (1999) 38:985; 82. J Biol Chem (1996) 271:10004; 83. J Biol Chem (1997) 272:18834; 84. J Biol Chem (1996) 271:19794; 85. Biochem Biophys Res Commun (1972) 46:597; 86. Science (1972) 175:646; 87. Biochemistry (1988) 27:3812; 88. J Biol Chem (2008) 283:19379; 89. J Biol Chem (1993) 268:8683; 90. J Cell Biol (1988) 106:1553; 91. J Biol Chem (1984) 259:11920; 92. Am J Physiol Lung Cell Mol Physiol (2000) 278:L1091; 93. Eur J Biochem (2003) 270:3479; 94. Biochim Biophys Acta (1973) 320:635; 95. Biochim Biophys Acta (1976) 453:293; 96. J Muscle Res Cell Motil (1992) 13:132; 97. Biochemistry (1992) 31:3930; 98. Biophys J (1992) 61:553; 99. Biochemistry (2006) 45:7237; 100. J Biol Chem (2006) 281:27471; 101. Br J Pharmacol (2003) 140:202; 102. Nat Methods (2007) 4:619; 103. Biophys J (1995) 68:78S-80S; 104. J Mol Biol (1992) 223:185; 105. Ann N Y Acad Sci (1982) 402:478; 106. Biophys J (1994) 67:2436; 107. Biophys J (1994) 67:1933; 108. J Biol Chem (1995) 270:23966; 109. Biophys J (1994) 66:1115; 110. J Biolumin Chemilumin (1994) 9:29; 111. Methods Cell Biol (1989) 31:63; 112. Annu Rev Physiol (1990) 52:857; 113. J Neurosci Methods (2004) 132:81; 114. J Physiol (1999) 519 Pt 3:723; 115. Mol Pharmacol (1999) 56:1171; 116. J Neurochem (1993) 61:1657; 117. Biochemistry (1989) 28:3989.








DATA TABLE 17.3 PROBES FOR PROTEIN KINASES, PROTEIN PHOSPHATASES AND NUCLEOTIDE-BINDING PROTEINSCat. No. MW Storage Soluble Abs EC Em Solvent NotesA1048 700.30 FF,D,LL H2O 259 18,000 none MeOH 1, 2, 3A1049 760.35 FF,D,LL H2O 351 4400 none H2O 1, 2A7056 614.44 FF,D,LL H2O 259 15,000 none MeOH 1, 2, 3A12410 933.30 FF,L H2O 505 54,000 514 H2O 4, 5A12412 1117.18 FF,L H2O 323 4200 461 pH 7 4, 5, 6A22184 878.28 FF,L H2O 504 68,000 514 pH 7 4, 5A22352 1065.43 FF,L H2O 591 55,000 620 pH 7 4, 5A22359 963.47 FF,L H2O 592 57,000 621 pH 7 4, 5A22362 ~2050 FF,L H2O 648 246,000 667 pH 7 4, 5A35775 1162.23 FF,L H2O 493 71,000 517 pH 7 4, 5A35777 ~1700 FF,L H2O 649 246,000 666 pH 7 4, 5B7469 784.70 F,D,L DMSO 504 79,000 511 MeOHB22356 932.31 FF,L H2O 504 68,000 514 H2O 4, 5B22358 1018.97 FF,L H2O 260 27,000 none pH 7D1037 524.38 F,D,LL DMSO 338 6100 none MeOH 1, 2E23691 619.13 FF H2O 265 5000 411 pH 7 5G12411 949.30 FF,L H2O 504 68,000 511 H2O 4, 5, 7G22183 894.28 FF,L H2O 504 68,000 510 pH 7 4, 5, 7G22351 1081.43 FF,L H2O 591 56,000 620 pH 7 4, 5, 7G22360 1005.75 FF,L H2O 504 68,000 508 pH 7 4, 5, 7G35778 905.29 FF,L H2O 505 68,000 512 pH 7 4, 5, 7G35779 865.28 FF,L H2O 511 520 pH 7 4, 5, 7G35780 1153.60 FF,L H2O 591 621 pH 7 4, 5, 7H7476 504.45 F,D,L DMSO, DMF 591 37,000 594 EtOHM12414 620.32 FF,L H2O 356 5700 447 pH 8 4, 5, 8M12415 722.28 FF,L H2O 356 5700 448 pH 7 4, 5, 8M12416 604.32 FF,L H2O 356 5800 448 pH 7 4, 5, 8M12417 706.28 FF,L H2O 356 5800 447 pH 7 4, 5, 8M22353 721.29 FF,L H2O 357 5700 447 pH 8 4, 5, 8M22354 705.29 FF,L H2O 357 5800 447 pH 8 4, 5, 9T7601 682.26 FF,L H2O 408 26,000 none pH 8 4, 5, 9T7602 784.22 FF,L H2O 408 26,000 none pH 8 4, 5, 9T7624 579.29 F,L H2O 408 26,000 none pH 8 4, 5, 9For de�nitions of the contents of this data table, see “Using The Molecular Probes® Handbook” in the introductory pages.Notes

1. Caged nucleotide esters are free of contaminating free nucleotides when initially prepared. However, some decomposition may occur during storage.2. All photoactivatable probes are sensitive to light. They should be protected from illumination except when photolysis is intended.3. This compound has weaker visible absorption at >300 nm but no discernible absorption peaks in this region.4. The molecular weight (MW) of this product is approximate because the degree of hydration and/or salt form has not been conclusively established.5. This product is supplied as a ready-made solution in the solvent indicated under "Soluble."6. QY = 0.63 in 50 mM Tris, pH 8.0. Fluorescence shifts to longer wavelengths (Em ~475 nm) on enzymatic cleavage of the α–β phosphoryl bond. (Biochem Biophys Res Commun (1978) 81:35, J Biol

Chem (1979) 254:12069)7. Fluorescence of BODIPY® dye–labeled guanosine derivatives is generally weak due to base-speci�c intramolecular quenching. (Anal Biochem (2001) 291:109)8. Fluorescence quantum yields of MANT nucleotides are environment-dependent. In H2O, QY is ~0.2. (Biochim Biophys Acta (1983) 742:496)9. Trinitrophenyl nucleotides are in fact very weakly �uorescent in water (Em ~560 nm). Fluorescence is blue-shifted and more intense in organic solvents (DMSO, EtOH) and when bound to pro-

teins (Em ~540 nm). Absorption spectrum also has a second, less intense peak at about 470 nm. (Biochim Biophys Acta (1982) 719:509)







PRODUCT LIST 17.3 PROBES FOR PROTEIN KINASES, PROTEIN PHOSPHATASES AND NUCLEOTIDE-BINDING PROTEINSCat. No. Product Quantity

A22359 adenosine 5’-diphosphate, BODIPY® TR 2’-(or-3’)-O-(N-(2-aminoethyl)urethane), disodium salt (BODIPY® TR ADP) *5 mM in bu�er* 100 µLA7056 adenosine 5’-diphosphate, P2-(1-(2-nitrophenyl)ethyl) ester, monopotassium salt (NPE-caged ADP) 5 mgA22184 adenosine 5’-O-(3-thiotriphosphate), BODIPY® FL thioester, sodium salt (BODIPY® FL ATP-γ-S, thioester) *5 mM in bu�er* 50 µLA22362 adenosine 5’-triphosphate, Alexa Fluor® 647 2’-(or-3’)-O-(N-(2-aminoethyl)urethane), hexa(triethylammonium) salt (Alexa Fluor® 647 ATP) *5 mM in bu�er* 100 µLA12410 adenosine 5’-triphosphate, BODIPY® FL 2’-(or-3’)-O-(N-(2-aminoethyl)urethane), trisodium salt (BODIPY® FL ATP) *5 mM in bu�er* 100 µLA22352 adenosine 5’-triphosphate, BODIPY® TR 2’-(or-3’)-O-(N-(2-aminoethyl)urethane), trisodium salt (BODIPY® TR ATP) *5 mM in bu�er* 100 µLA1048 adenosine 5’-triphosphate, P3-(1-(2-nitrophenyl)ethyl) ester, disodium salt (NPE-caged ATP) 5 mgA1049 adenosine 5’-triphosphate, P3-(1-(4,5-dimethoxy-2-nitrophenyl)ethyl) ester, disodium salt (DMNPE-caged ATP) 5 mgA12412 adenosine 5’-triphosphate, P3-(5-sulfo-1-naphthylamide), tetra(triethylammonium) salt (ATP γ-AmNS) *5 mM in bu�er* 400 µLA35775 Alexa Fluor® 488 8-(6-aminohexyl)aminoadenosine 3’,5’-cyclicmonophosphate, bis(triethylammonium) salt (Alexa Fluor® 488 cAMP) *5 mM in bu�er* 100 µLA35777 Alexa Fluor® 647 8-(6-aminohexyl)aminoadenosine 3’,5’-cyclicmonophosphate, tetra(triethylammonium) salt (Alexa Fluor® 647 cAMP) *5 mM in bu�er* 100 µLA35725 Antibody Beacon™ Tyrosine Kinase Assay Kit *400 assays* 1 kitA6442 anti-synapsin I (bovine), rabbit IgG fraction *a�nity puri�ed* 10 µgB22358 2’-(or-3’)-O-(4-benzoylbenzoyl)adenosine 5’-triphosphate, tris(triethylammonium) salt (BzBzATP) *5 mM in bu�er* 2 mLB7469 BODIPY® FL forskolin 100 µgB22356 2’-(or-3’)-O-(BODIPY® FL)-β:γ-imidoadenosine 5’-triphosphate, trisodium salt (BODIPY® FL AMPPNP) *5 mM in bu�er* 50 µLC10558 cAMP Chemiluminescent Immunoassay Kit *10-plate size* 1 kitC10557 cAMP Chemiluminescent Immunoassay Kit *2-plate size* 1 kitD1037 4,5-dimethoxy-2-nitrobenzyl adenosine 3’,5’-cyclicmonophosphate (DMNB-caged cAMP) 5 mgE23691 1,N6-ethenoadenosine 5’-triphosphate (ε-ATP) *5 mM in bu�er* 2 mLG22360 guanosine 5’-diphosphate, BODIPY® FL 2’-(or-3’)-O-(N-(2-aminoethyl)urethane), bis(triethylammonium) salt (BODIPY® FL GDP) *5 mM in bu�er* 100 µLG35778 guanosine 5’-O-(3-iminotriphosphate), BODIPY® FL ethylamide, sodium salt (BODIPY® FL GTP-γ-NH, amide) *1 mM in bu�er* 100 µLG35779 guanosine 5’-O-(3-thiotriphosphate), BODIPY® 515/530 thioester, sodium salt (BODIPY® 515/530 GTP-γ-S, thioester) *1 mM in bu�er* 100 µLG22183 guanosine 5’-O-(3-thiotriphosphate), BODIPY® FL thioester, sodium salt (BODIPY® FL GTP-γ-S, thioester) *5 mM in bu�er* 50 µLG35780 guanosine 5’-O-(3-thiotriphosphate), BODIPY® TR thioester, sodium salt (BODIPY® TR GTP-γ-S, thioester) *1 mM in bu�er* 100 µLG12411 guanosine 5’-triphosphate, BODIPY® FL 2’-(or-3’)-O-(N-(2-aminoethyl)urethane), trisodium salt (BODIPY® FL GTP) *5 mM in water* 100 µLG22351 guanosine 5’-triphosphate, BODIPY® TR 2’-(or-3’)-O-(N-(2-aminoethyl)urethane), trisodium salt (BODIPY® TR GTP) *5 mM in water* 100 µLH7476 hypericin 1 mgM12416 2’-(or-3’)-O-(N-methylanthraniloyl)adenosine 5’-diphosphate, disodium salt (MANT-ADP) *5 mM in bu�er* 400 µLM12417 2’-(or-3’)-O-(N-methylanthraniloyl)adenosine 5’-triphosphate, trisodium salt (MANT-ATP) *5 mM in bu�er* 400 µLM12414 2’-(or-3’)-O-(N-methylanthraniloyl)guanosine 5’-diphosphate, disodium salt (MANT-GDP) *5 mM in bu�er* 400 µLM12415 2’-(or-3’)-O-(N-methylanthraniloyl)guanosine 5’-triphosphate, trisodium salt (MANT-GTP) *5 mM in bu�er* 400 µLM22354 2’-(or-3’)-O-(N-methylanthraniloyl)-β:γ-imidoadenosine 5’-triphosphate, trisodium salt (MANT-AMPPNP) *5 mM in bu�er* 50 µLM22353 2’-(or-3’)-O-(N-methylanthraniloyl)-β:γ-imidoguanosine 5’-triphosphate, trisodium salt (MANT-GMPPNP) *5 mM in bu�er* 50 µLP13235 polymyxin B, BODIPY® FL conjugate, tri�uoroacetic acid salt *mixed species* 100 µgP13238 polymyxin B, dansyl conjugate, tri�uoroacetic acid salt *mixed species* 100 µgP13236 polymyxin B, Oregon Green® 514 conjugate, tri�uoroacetic acid salt *mixed species* 100 µgP33706 Pro-Q® Diamond Phosphoprotein/Phosphopeptide Microarray Stain Kit 1 kitR33700 RediPlate™ 96 EnzChek® Serine/Threonine Phosphatase Assay Kit *one 96-well microplate* 1 kitR22067 RediPlate™ 96 EnzChek® Tyrosine Phosphatase Assay Kit *one 96-well microplate* 1 kitT7601 2’-(or-3’)-O-(trinitrophenyl)adenosine 5’-diphosphate, disodium salt (TNP-ADP) *5 mg/mL in water* 2 mLT7624 2’-(or-3’)-O-(trinitrophenyl)adenosine 5’-monophosphate, sodium salt (TNP-AMP) *5 mg/mL in bu�er* 2 mLT7602 2’-(or-3’)-O-(trinitrophenyl)adenosine 5’-triphosphate, trisodium salt (TNP-ATP) *5 mg/mL in bu�er* 2 mL







Section 17.4 Probes for Lipid Metabolism and Signaling

17.4 Probes for Lipid Metabolism and Signaling

Lipids and lipid metabolites are abundant in cells and have both a structural function and a role in cell regulation. Phospholipases, in particular, play an important part in cellular signal-ing processes via the generation of second messengers such as diacylglycerols, arachidonate and inositol 1,4,5-triphosphate 1–4 (Ins 1,4,5-P3, I3716; Section 17.2). In addition, phospholipase A2 activation is a key step in in�ammation processes, and phospholipase A2 plays major roles in bacterial virulence and in the pathogenesis of acute respiratory distress syndrome 5–7 (ARDS), making this class of enzymes important therapeutic targets.8,9

Phospholipases are classi�ed according to the cleavage site on the phospholipid substrate (Figure 17.4.1). �ere are at least three types of �uorescence-based phospholipase detection methods: 10

• Continuous methods, which permit direct �uorometric monitoring of enzymatic activity using self-quenching or excimer-forming probes

• Methods that continuously detect non�uorescent product formation from natural phospho-lipids, such as detection of fatty acids with our ADIFAB reagent or enzyme-coupled detec-tion of choline with our Amplex® Red Phospholipase Assay Kits

• Discontinuous methods, which require resolution of �uorescent substrates and products by TLC, HPLC or other separation techniques

Table 17.3 summarizes Molecular Probes® products for �uorescence-based phospholipase assays. Other applications for our wide range of �uorescent phospholipids are described in Chapter 13.

Figure 17.4.1 Cleavage speci�cities of phospholipas-es. R1 and R2 are typically saturated or unsaturated aliphatic groups. The polar head group R3 can be choline, ethanol-amine, glycerol, inositol, inositol phosphate, serine or other alcohols.

PLA1

PLA2

OCH2OCH

CH2O

CR1

CR2

O

O P O R3O

O_

PLD

PLC

Table 17.3 Fluorescence-based phospholipase assays.

Phospholipase * Probes Assay Principle Detection Method

A1 A10070, E10219, E10221 Intramolecular self-quenching Fluorescence increase at ~530 nm

A1, A2 B7701 Intramolecular self-quenching Fluorescence increase at ~515 nm 1

A1, A2 B3781, B3782 Intramolecular excimer formation Emission ratio 380/470 nm 2–4

A1, A2 A3880 Free fatty acid sensor Emission ratio 432/v505 nm 5–7

A2 A10072, E10217, E10218 Intramolecular �uorescence resonance energy transfer (FRET)

Fluorescence increase at ~515 nm or increase in emission ratio at 515/575 nm

A2 D23739 Intramolecular self-quenching Fluorescence increase at ~515 nm 8

A2 N3786, N3787 Intermolecular self-quenching Fluorescence increase at ~530 nm 9,10

A2 H361, H3809 Intermolecular excimer formation Emission ratio 380/470 nm 11–13

A2 D3803 Release of a �uorescent fatty acid TLC or �uorescence image scanner 14

A2, C, D D3771 Formation of a �uorescent O-alkylglycerol derivative TLC or HPLC 15–17

A2, C, D H361 Quenching by a disul�de-polymerized lipid matrix Fluorescence increase at ~380 nm 18,19

C A12218 Peroxidase-linked detection of phosphocholine Conversion of the non�uorescent Amplex® Red reagent to �uorescent resoru�n 20–22

C E10215, E10216 Release of dye-labeled diacylglycerol Fluorescence increase at 516 nm, with potential interference from phospholipase A2 and phospholipase D activity

D A12219 Peroxidase-linked detection of choline Conversion of the non�uorescent Amplex® Red reagent to �uorescent resoru�n 20,22,23

PAP † D3805 Release of dye-labeled diacylglycerol HPLC 24

* Phospholipase speci�city: A1, A2, C or D (see Section 17.4 for cleavage speci�cities). † PAP = phosphatidic acid phosphohydrolase.1. J Biol Chem (1992) 267:21465; 2. Biochemistry (1993) 32:583; 3. Anal Biochem (1981) 116:553; 4. Biochim Biophys Acta (1994) 1192:132; 5. Anal Biochem (1995) 229:256; 6. Biochem J (1994) 298:23; 7. J Biol Chem (1992) 267:23495; 8. Anal Biochem (1999) 276:27; 9. Lipids (1989) 24:691; 10. Biochem Biophys Res Commun (1984) 118:894; 11. Anal Biochem (2006) 359:280; 12. Chem Phys Lipids (1990) 53:129; 13. Anal Biochem (1989) 177:103; 14. J Biol Chem (1999) 274:19338; 15. Eukaryot Cell (2009) 8:1094; 16. Anal Biochem (1994) 218:136; 17. Biochem J (1995) 307:799; 18. Anal Biochem (1994) 221:152; 19. J Biol Chem (1995) 270:263; 20. Proc Natl Acad Sci U S A (2004) 101:9745; 21. Mol Pharmacol (2000) 57:1142; 22. Proc SPIE-Int Soc Opt Eng (2000) 3926:166; 23. J Biol Chem (2002) 277:45592; 24. Anal Biochem (2008) 374:291.







Phospholipase A1 and A2 Assays�e importance of phospholipases in cellular signaling, lipid me-

tabolism, in�ammatory responses and pathological disorders related to these processes has stimulated demand for �uorescence-based enzyme activity monitoring methods. Several of the �uorogenic phospholipase A substrates described here are designed to provide continuous moni-toring of phospholipase A activity in puri�ed enzyme preparations, cell lysates and live cells; applications of some of these substrates extend as far as in vivo small animal imaging.11–13 �e phospholipase A substrates are generally dye-labeled phospholipids of two types—glycerophospho-cholines with BODIPY® dye–labeled sn-1 or sn-2 (or both) acyl or alkyl chains and glycerophosphoethanoloamines with BODIPY® dye–labeled acyl chains and dinitrophenyl quencher–modi�ed head groups (Figure 17.4.2). �ese structural variations determine speci�city for phospholi-pase A1 (which hydrolyzes the sn-1 ester linkage between phospholipids and fatty acids) versus phospholipase A2 (which hydrolyzes the sn-2

Figure 17.4.2 Mechanism of phospholipase activity–linked �uorescence enhancement responses of bis-BODIPY® FL C11-PC (B7701) and PED6 (D23739). Note that enzymatic cleavage of bis-BODIPY® FL C11-PC yields two �uorescent products, whereas cleavage of PED6 yields only one.

+

Fluorescent lysophospholipid

Quenched substrate

+

C

O

(CH2)10H3C

H3C

F F

NB

N

C

O

(CH2)10H3C

H3C

F F

NB

N CH2O

OCH

OCH2

OCH2CH2N(CH3)3O

O

P

(bis-BODIPY FL C11-PC)

Fluorescent fatty acid (BODIPY FL C11 (D-3862))

Phospholipase A2

C

O

(CH2)10H3C

H3C

F F

NB

N

OH

+P

O

O

OCH2CH2N(CH3)3

OCH2HOCH

CH2O

NB

N

FF

H3C

H3C (CH2)10

O

C

NB

N

F F

+

Non�uorescent lysophospholipid

Quenched substrate (PED6)

NO2

O2N

(CH2)5NH

O

COCH2CH2NH

O

O

P

CH3(CH2)14

O

C

CH2O

OCH

OCH2C

O

(CH2)4H3C

H3C

CH3(CH2)14

O

C OCH2HOCH

CH2O P

O

O

OCH2CH2NH C

O

(CH2)5NH

O2N

NO2

Phospholipase A2

Fluorescent fatty acid (BODIPY FL C5 (D-3834))

C

O

(CH2)4H3C

H3C

F F

NB

N

OH

Figure 17.4.3 PED-A1 (N-((6-(2,4-DNP)amino)hexanoyl)-1-(BODIPY® FL C5)-2-hexyl-sn-glycero-3-phosphoethanolamine, A10070).

C OCH2

CH3(CH2)� OCH

CH2O P OCH2CH2NHO

OH

(CH2)�

O

C (CH2)�NHO

O2N

NO2

��H3C

N�

N

H3C

ester linkage between phospholipids and fatty acids, Figure 17.4.1), and the �uorescence response associated with enzymatic cleavage of the substrate (Table 17.3).

PED-A1 Phospholipase A1 SubstratePED-A1 (A10070, Figure 17.4.3) is a �uorogenic substrate designed

to provide speci�c, real-time monitoring of phospholipase A1 activity in puri�ed enzyme preparations, cell lysates and live cells.14,15 PED-A1 is comprised of a dinitrophenyl quencher–modi�ed glycerophospho-ethanolamine head group and a green-�uorescent BODIPY® FL dye–labeled acyl chain at the sn-1 position. Upon cleavage by phospholipase A1, PED-A1 exhibits an increase in green �uorescence (measured at excitation/emission = 488/530 nm). Phospholipase A1 speci�city is im-parted by the placement of the BODIPY® FL acyl chain in the sn-1 posi-tion and by incorporation of an acyl group with an enzyme-resistant (noncleavable) ether linkage in the sn-2 position.








EnzChek® Phospholipase A1 Assay Kit�e EnzChek® Phospholipase A1 Assay Kit (E10219, E10221) provides a simple, �uorometric

method for continuous monitoring of phospholipase A1 activity based on the phospholipase A1–speci�c PED-A1 substrate (A10070, described above). �e EnzChek® Phospholipase A1 Assay Kit can detect phospholipase A1 activity at 0.04 U/mL or lower (Figure 17.4.4). �is microplate-based assay is well suited for rapid and direct analysis of phospholipase A1 in puri�ed enzyme preparations and cell lysates using automated instrumentation, as well as for characterizing phospholipase A1 inhibitors.

Each EnzChek® Phospholipase A1 Assay Kit (2-plate size, E10219; 10-plate size, E10221) provides:

• PED-A1 phospholipase A1 substrate• Phospholipase A1 (Lecitase Ultra)• Concentrated phospholipase A1 reaction bu�er• Dioleoylphosphatidylcholine (DOPC)• Dioleoylphosphatidylglycerol (DOPG)• Dimethylsulfoxide (DMSO)• Detailed assay protocols

�e 2-plate assay kit provides su�cient reagents for 200 reactions in 96-well microplates at a volume of 100 µL per well or 800 reactions using low-volume 384-well microplates at a volume of ≤25 µL per well. �e 10-plate assay kit provides su�cient reagents for 1000 reactions in 96-well microplates at a volume of 100 µL per well or 4000 reactions using low-volume 384-well micro-plates at a volume of ≤25 µL per well.

Red/Green BODIPY® PC-A2 Ratiometric Phospholipase A2 SubstrateRed/Green BODIPY® PC-A2 (A10072, Figure 17.4.5) is a ratiometric �uorogenic substrate

designed to provide selective, real-time monitoring of phospholipase A2 activity in puri�ed enzyme preparations, cell lysates and live cells. Cleavage of the BODIPY® FL pentanoic acid substituent at the sn-2 position results in decreased quenching by �uorescence resonance en-ergy transfer (FRET) of the BODIPY® 558/568 dye attached at the sn-1 position. �us, upon cleavage by phospholipase A2, Red/Green BODIPY® PC-A2 exhibits an increase in BODIPY® FL �uorescence, detected from 515–545 nm (Figure 17.4.6). �e FRET-sensitized BODIPY® 558/568 �uorescence signal is expected to show a reciprocal decrease; in practice, however, this longer-wavelength �uorescence may show a decrease or a slight increase, depending on the formulation of the substrate and the instrument wavelength settings. �e ratiometric detection mode of this substrate (emission intensity ratio at 515⁄575 nm with excitation at ~460 nm) allows measure-ments of phospholipase A2 activity that are essentially independent of instrumentation and assay conditions. �e dual-emission properties of this substrate also provide the capacity to localize the lysophospholipid and fatty acid products of the phospholipase A2 cleavage via their distinct spectroscopic signatures in imaging experiments.

EnzChek® Phospholipase A2 Assay Kit�e EnzChek® Phospholipase A2 Assay Kit (E10217, E10218) provides a simple, �uoromet-

ric method for continuous monitoring of phospholipase A2 activity based on the phospholipase A2–selective Red/Green BODIPY® PC-A2 (A10072, described above). �is phospholipase A2 as-say can be used in an intensity-based detection mode, by following the �uorescence increase at

Figure 17.4.4 Detection of phospholipase A1 (PLA1) using the EnzChek® Phospholipase A1 Assay Kit (E10219, E10221). PLA1 reactions were run at ambient temperature with liposomes for 30 minutes according to the assay pro-tocol provided, and �uorescence emission was measured using 460 nm excitation on a Spectra Max M5 (Molecular Devices). Background �uorescence determined for the no-enzyme control reaction has been subtracted.

PLA1 concentration (U/mL)

Fluo

resc

ence

inte

nsity

5430

1,000

2.000

3,000

4,000

5,000

6,000

7,000

8,000

0 1 2

5001,0001,5002,0002,5003,0003,500

0 0.2 0.4 0.6 0.8 1 1.20

Figure 17.4.6 Fluorescence emission spectra (excitation at 480 nm) of Red/Green BODIPY® PC-A2 phospholipase A2 substrate (A10072) incorporated in liposomes with addition of bee venom phospholipase A2 at ambient temperature.

Emission (nm)

Inte

nsity

490.0

0

100

200

300

400

500

600

700

800

900

1,000

500 620 640600520 540 560 580650.5

66 min21 min11 min6 min1 min0 min

Figure 17.4.5 Red/Green BODIPY® PC-A2 (1-O-(6-BODIPY® 558/568-aminohexyl)-2-BODIPY® FL C5-sn-glycero-3-phospho choline, A10072).

��N

�N

CH2CH2

��N

�N

(CH2)� C OCHO

S

CH2O P OCH2CH2N(CH3)3

O

O

H3C

H3C

C NH(CH2)6

OOCH2







~515 nm, or in a ratiometric-based detection mode, by following the changes in the emission intensity ratio at 515/575 nm with excitation at ~460 nm (Figure 17.4.7). �e EnzChek® Phospholipase A2 Assay Kit can detect bee venom phospholipase A2 activity at 0.05 U/mL or lower (Figure 17.4.7). �is microplate-based assay is well suited for rapid and direct analysis of phospholipase A2 in puri�ed enzyme preparations and cell lysates using automated instrumentation, as well as for character-izing phospholipase A2 inhibitors.

Each EnzChek® Phospholipase A2 Assay Kit (2-plate size, E10217; 10-plate size, E10218) provides:

• Red/Green BODIPY® PC-A2 phospholipase A2 substrate• Phospholipase A2 from honey bee venom• Concentrated phospholipase A2 reaction bu�er• Dioleoylphosphatidylcholine (DOPC)• Dioleoylphosphatidylglycerol (DOPG)• Dimethylsulfoxide (DMSO)• Detailed assay protocols

�e 2-plate assay kit provides su�cient reagents for 200 reactions in 96-well microplates at a volume of 100 µL per well or 800 reactions us-ing low-volume 384-well microplates at a volume of ≤25 µL per well. �e 10-plate assay kit provides su�cient reagents for 1000 reactions in 96-well microplates at a volume of 100 µL per well or 4000 reactions using low-volume 384-well microplates at a volume of ≤25 µL per well.

PED6 Phospholipase A2 SubstratePED6 (D23739, Figure 17.4.8) is a �uorogenic substrate for phos-

pholipase A2 incorporating a BODIPY® FL dye–labeled sn-2 acyl chain and a dinitrophenyl quencher–labeled head group 16 (Figure 17.4.2). Cleavage of the dye-labeled acyl chain by phospholipase A2 eliminates the intramolecular quenching e�ect of the dinitrophenyl group, resulting in a corresponding �uorescence increase. Continuous kinetic assays show PED6 to be a good substrate for both secreted and cytosolic phospholipase A2 and platelet-activating factor acetyl-hydrolase.16 PED6 has been used by Steven Farber and co-workers for in vivo analysis of intestinal lipid metabolism in zebra�sh larvae as a basis for identifying and screening mutant phenotypes 12,13,17 (Figure 17.4.9). PED6 is also useful for high-throughput screening of potential phospholipase A2 inhibitors or activators.

Other BODIPY® Dye Phospholipase A Substrates�e bis-BODIPY® phospholipase A substrate—bis-BODIPY® FL

glycerophosphocholine (bis-BODIPY® FL C11-PC, B7701)—has been speci�cally designed to allow continuous monitoring of phospho-lipase A action and to be spectrally compatible with argon-ion laser excitation sources.18 When this probe is incorporated into cell mem-branes, the proximity of the BODIPY® FL �uorophores on adjacent phospholipid acyl chains causes �uorescence self-quenching (Figure 17.4.2). Separation of the �uorophores upon hydrolytic cleavage of one of the acyl chains by either phospholipase A1 or A2 results in increased �uorescence. Bis-BODIPY® FL C11-PC has been developed in collabora-tion with Elizabeth Simons, who has successfully employed it for �ow cytometric detection of phospholipase A activity in neutrophils.19 More recently, bis-BODIPY® FL C11-PC has been used to detect phospholipase A2 activation induced by tumor necrosis factor (TNF) 20 and for high-throughput assays of endothelial lipase, a critical determinant of HDL cholesterol levels.21,22

Figure 17.4.7 Detection of phospholipase A2 (PLA2) using the EnzChek® Phospholipase A2 Assay Kit (E10217, E10218). PLA2 reactions were run at ambient temperature with liposomes for 10 minutes according to the assay protocol provided, and �uorescence emission was measured using 460 nm excitation on a Spectra Max® M5 (Molecular Devices). Background �uorescence determined for the no-enzyme control reaction has been subtracted. Top panel shows ratiometric-based (515/575 nm) detection mode; bottom panel shows intensity-based (515 nm channel) detection mode. Background �uorescence determined for the no-enzyme control reaction has been subtracted for each value.

PLA2 concentration (U/mL)0 321 54

Fluo

resc

ence

inte

nsity

500

1,000

1,500

2,000

2,500

3,000

3,500

0

0200400

1,2001,400

1,000800600

1,600

0 0.2 0.6 0.8 10.4

PLA2 concentration (U/mL)0 3 421 5

Fluo

resc

ence

rat

io

1

2

3

4

5

6

7

0

0

1.5

0.51.0

4.04.5

3.5

2.53.0

2.0

5.0

0 0.2 0.6 0.8 10.4

Figure 17.4.8 N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-2-(4,4-di�uoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (PED6) (D23739).

C OCH2

OCH

CH2O P OCH2CH2NHO

O

CH3(CH2)��

O

C (CH2)�NHO

O2N

NO2

��N

�N

(CH2)� C

H3C

H3CO

(CH3CH2)3NH

Figure 17.4.9 Imaging of lipid digestion pathways in zebra�sh (Danio rerio) using the �uoro-genic phospholipase A2 substrate PED6 (D23739). A zebra�sh larva (5 days post-fertilization) was incubated with PED6 for 2 hours. Localized �uorescence in the gallbladder and intestinal lumen results from endogenous lipase activity and rapid transport of the substrate cleav-age products through the intestinal and hepatobiliary systems. The image was provided by Steven A. Farber, Thomas Je�erson University.








Speci�city for phospholipase A2 versus phospholipase A1 can be obtained using phospho-lipids with nonhydrolyzable, ether-linked alkyl chains in the sn-1 position. A 1-O-alkyl–substi-tuted phospholipid containing the BODIPY® FL �uorophore (D3771, Figure 17.4.10) is a useful substrate for a phospholipase A2–speci�c chromatographic assay.23

�e singly labeled BODIPY® phospholipase A2 substrate—β-BODIPY® FL C5-HPC (D3803)—has been used to quantitatively delineate a discontinuous increase of Ca2+-dependent cytosolic phospholipase A2 (cPLA2) activity during zebra�sh embryogenesis. �e analytical method devel-oped for this study uses a �uorescence image scanner to quantitatively detect the free BODIPY® FL dye–labeled fatty acid generated by the action of cPLA2

24 (Figure 17.4.11).

Bis-Pyrenyl Phospholipase A SubstratesOur bis-pyrenyl phospholipase A probes (B3781, B3782) both emit at ~470 nm, indicat-

ing that their adjacent pyrene �uorophores (Figure 17.4.12) form excited-state dimers (Figure 17.4.13). Phospholipase A–mediated hydrolysis separates the �uorophores, which then emit as monomers at ~380 nm.25 �ese substrates have proven to be e�ective phospholipase A2 sub-strates in model membrane systems (Table 17.3); however, it has been reported that 1,2-bis-(1-pyrenebutanoyl)-sn-glycero-3-phosphocholine (B3781) is highly resistant to degradation by phos-pholipases in human skin �broblasts.26 1,2-bis-(1-pyrenebutanoyl)-sn-glycero-3-phosphocholine has been used in a sensitive, continuous assay for lecithin:cholesterol acyltransferase 27,28 (LCAT).

Singly Labeled Pyrenyl and NBD Phospholipase A2 SubstratesPhospholipase A2 activity has also been measured using phospholipids labeled with a single

pyrene (H361, Figure 17.4.14; H3809, Figure 17.4.15) or NBD (N3786; N3787, Figure 17.4.16) �uo-rophore (Table 17.3). Because only the sn-2 phospholipid acyl chain is labeled, these probes can discriminate between phospholipase A2 and phospholipase A1 activity. To obtain a direct �uo-rescence response to enzymatic cleavage, su�cient phospholipid must be loaded into membranes to cause either intermolecular self-quenching (NBD-acyl phospholipids) or excimer formation 29 (pyreneacyl phospholipids). Pyrene-labeled acidic phospholipids—particularly the phosphoglyc-erol derivative 30,31 (H3809)—are preferred as substrates by pancreatic and intestinal phospho-lipase A2, whereas labeled phosphocholine (H361, Figure 17.4.14) is preferred by phospholipase A2 from snake venom.32

ADIFAB Indicator: A Di�erent View of Phospholipase A Activity�e ADIFAB fatty acid indicator (A3880, Figure 17.4.17) functions as a �uorescent sensor for

the free fatty acid cleavage products of phospholipases.33–36 It does not require membrane loading

Figure 17.4.10 2-decanoyl-1-(O-(11-(4,4-di�uoro-5,7-dimeth-yl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)undecyl)-sn-glycero-3-phosphocholine (D3771).

Figure 17.4.11 Assay of cytoplasmic phospholipase A2 (cPLA2) using β-BODIPY® FL C5-HPC (D3803) as a sub-strate. The substrate was incubated in enzyme-free assay bu�er (lane 1), with secreted PLA2 (from Naja mossambica; lane 2) or with puri�ed human recombinant cPLA2 (lane 3). Cleavage products were separated by thin-layer chro-matography in chloroform/methanol/acetic acid/water (50:30:8:4) and were subsequently analyzed using a �uores-cence image scanner. Both phospholipases liberated �uores-cent BODIPY® FL dye–labeled fatty acids (FFA) by cleavage of the substrate at the sn-2 acyl bond. Figure reproduced with permission from J Biol Chem (1999) 274:19338.

1 2 3Blan

ksP

LA2

cPLA

2

FFA product

Phospholipid substrate

Figure 17.4.12 1,2-bis-(1-pyrenebutanoyl)-sn-glycero-3-phosphocholine (B3781).

Figure 17.4.13 Excimer formation by pyrene in etha-nol. Spectra are normalized to the 371.5 nm peak of the monomer. All spectra are essentially identical below 400 nm after normalization. Spectra are as follows: 1) 2 mM pyrene, purged with argon to remove oxygen; 2) 2 mM pyrene, air-equilibrated; 3) 0.5 mM pyrene (argon-purged); and 4) 2 µM pyrene (argon-purged). The monomer-to-excimer ratio (371.5 nm/470 nm) is dependent on both pyrene concentra-tion and the excited-state lifetime, which is variable because of quenching by oxygen.

Fluo

resc

ence

em

issi

on

Wavelength (nm)350 450400 500 550 600

1

2

3

4

Figure 17.4.14 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (β-py-C10-HPC) (H361).

Figure 17.4.15 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphoglycerol, ammonium salt (β-py-C10-PG) (H3809).







and can be used to monitor hydrolysis of natural (rather than synthetic) substrates. Assaying ly-sophospholipase activity with ADIFAB yields sensitivity comparable to radioisotopic methods.37 Richieri and Kleinfeld have described a methodology for using the ADIFAB reagent to measure the activity of phospholipase A2 on cell and lipid-vesicle membranes; their assay is capable of detecting hydrolysis rates as low as 10–12 mole/minute.36

Phospholipase C AssaysEnzChek® Direct Phospholipase C Assay Kit

�e EnzChek® Direct Phospholipase C Assay Kit (E10215, E10216) provides a simple and robust microplate-based method for monitoring phosphatidylcholine-speci�c phospholipase C (PC-PLC) activity in puri�ed enzyme preparations. PC-PLC plays a crucial role in many cell signaling pathways involved in apoptosis and cell survival, as well as in diseases as diverse as cancer and HIV. �is assay uses a proprietary substrate (glycerophosphoethanolamine with a dye-labeled sn-2 acyl chain) to detect PC-PLC activity. Substrate cleavage by PC-PLC releases dye-labeled diacylglycerol, which produces a positive �uorescence signal that can be measured continuously using a �uorescence microplate reader. �e reaction product has �uorescence ex-citation and emission maxima of 509 nm and 516 nm, respectively.

�e EnzChek® Direct Phospholipase C Assay Kit has been optimized using puri�ed PC-PLC from Bacillus cereus. �is assay may be amenable for use with cells and cell lysates, although the presence of phospholipase A2 or phospholipase D activity can potentially result in confound-ing signal enhancement. Using the EnzChek® Direct Phospholipase C Assay Kit with puri�ed enzyme from Bacillus cereus, we can typically detect as little as 10 mU/mL PC-PLC a�er one hour incubation at room temperature (Figure 17.4.18). �is kit is also useful for characterizing PC-PLC inhibition, and because it o�ers a direct measurement, the potential for false positives in a compound screen is reduced.

Each EnzChek® Direct Phospholipase C Assay Kit (2-plate size, E10215; 10-plate size, E10216) provides:

• Phosphatidylcholine-speci�c phospholipase C (PC-PLC) substrate• Phospholipase C from Bacillus cereus• Concentrated phospholipase C reaction bu�er• Phosphatidylcholine (lecithin)• Dimethylsulfoxide (DMSO)• Detailed assay protocols

�e 2-plate assay kit provides su�cient reagents for 200 reactions in 96-well microplates at a volume of 200 µL per well or 2000 reactions using low-volume 384-well microplates at a volume of 20 µL per well. �e 10-plate assay kit provides su�cient reagents for 1000 reactions in 96-well microplates at a volume of 200 µL per well or 10,000 reactions using low-volume 384-well mi-croplates at a volume of 20 µL per well.

Amplex® Red Phosphatidylcholine-Speci�c Phospholipase C Assay Kit�e Amplex® Red Phosphatidylcholine-Speci�c Phospholipase C Assay Kit (A12218) pro-

vides a sensitive method for continuously monitoring phosphatidylcholine-speci�c phospho-lipase C (PC-PLC) activity in vitro using a �uorescence microplate reader or �uorometer.38–40 In this enzyme-coupled assay, PC-PLC activity is monitored indirectly using the Amplex® Red reagent, a sensitive �uorogenic probe for H2O2 (Section 10.5). First, PC-PLC converts the phos-phatidylcholine (lecithin) substrate to form phosphocholine and diacylglycerol. A�er the action of alkaline phosphatase, which hydrolyzes phosphocholine to inorganic phosphate and choline, choline is oxidized by choline oxidase to betaine and H2O2. Finally, H2O2, in the presence of horseradish peroxidase, reacts with the Amplex® Red reagent in a 1:1 stoichiometry to generate the highly �uorescent product, resoru�n. Because resoru�n has absorption and �uorescence emission maxima of approximately 571 nm and 585 nm, respectively, there is little interference from auto�uorescence in most biological samples.

�e Amplex® Red Phosphatidylcholine-Speci�c Phospholipase C Assay Kit is potentially useful for detecting PC-PLC activity in cell extracts and for screening PC-PLC inhibitors. Experiments

Figure 17.4.17 Ribbon representation of the ADIFAB free fatty acid indicator (A3880). In the left-hand image, the fatty acid binding site of intestinal fatty acid–binding pro-tein (yellow) is occupied by a covalently attached acrylodan �uorophore (blue). In the right-hand image, a fatty acid molecule (gray) binds to the protein, displacing the �uoro-phore (green) and producing a shift of its �uorescence emis-sion spectrum. Image contributed by Alan Kleinfeld, FFA Sciences LLC, San Diego.

Figure 17.4.18 Detection of phosphatidylcholine-spe-ci�c phospholipase C (PC-PLC) using the EnzChek® Direct Phospholipase C Assay Kit (E10215, E10216). Triplicate samples of PC-PLC from Bacillus cereus were assayed at concentration of 7.8 mU/mL to 500 mU/mL per well in the presence of 1X PLC substrate and 200 μM lecithin in 1X PLC reaction bu�er. Reactions were incubated at room tem-perature for 60 minutes and �uorescence was measured using excitation/emission wavelengths of 490/520 nm. The inset represents a separate experiment and illustrates the linearity of �uorescence response at low levels of PC-PLC. The average variation of replicates (CV) was less than 3%. Background �uorescence determined for the no-enzyme control reaction has been subtracted.

0

200

400

600

800

1,000

0 100 200 300 400 500

PLC concentration (mU/mL)

Fluo

resc

ence

0

200

400

600

0 50 100 150

R2 = 0.9986

Figure 17.4.16 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phospho-choline (NBD C12-HPC) (N3787).








with puri�ed PC-PLC from Bacillus cereus indicate that the Amplex® Red Phosphatidylcholine-Speci�c Phospholipase C Assay Kit can detect PC-PLC levels as low as 0.2 mU/mL using a reaction time of one hour (Figure 17.4.19). One unit of PC-PLC is de�ned as the amount of enzyme that will liberate 1.0 micromole of water-soluble organic phosphorus from L-α-phosphatidylcholine per minute at pH 7.3 at 37°C.

Each Amplex® Red Phosphatidylcholine-Speci�c Phospholipase C Assay Kit includes:

• Amplex® Red reagent• Dimethylsulfoxide (DMSO)• Horseradish peroxidase (HRP)• H2O2 for use as a positive control• Concentrated reaction bu�er• Choline oxidase from Alcaligenes sp.• Alkaline phosphatase from calf intestine• L-α-Phosphatidylcholine (lecithin)• Phosphatidylcholine-speci�c phospholipase C from Bacillus cereus• Detailed protocols

Each kit provides su�cient reagents for approximately 500 assays using a �uorescence mi-croplate reader and a reaction volume of 200 µL per assay.

Bacillus cereus PI-PLCPhosphatidylinositol-speci�c phospholipase C (PI-PLC, EC 3.1.4.10) from Bacillus cereus

cleaves phosphatidylinositol (PI), yielding water-soluble D-myo-inositol 1,2-cyclic monophos-phate and lipid-soluble diacylglycerol.41 �is enzyme also functions to release enzymes that are linked to glycosylphosphatidylinositol (GPI) membrane anchors. We o�er highly puri�ed B. ce-reus PI-PLC (P6466), which has been used in studies of PI synthesis and export across the plasma membrane.42 PI-PLC generates diacylglycerols for PKC-linked signal transduction studies 43 and provides an e�cient means of releasing most GPI-anchored proteins from cell surfaces under conditions in which the cells remain viable.44,45

Phospholipase D AssaysAmplex® Red Phospholipase D Assay Kit

�e Amplex® Red Phospholipase D Assay Kit (A12219) provides a sensitive method for mea-suring phospholipase D (PLD) activity in vitro using a �uorescence microplate reader or �uorom-eter.38,40,46 In this enzyme-coupled assay, PLD activity is monitored indirectly using the Amplex® Red reagent (Section 10.5). First, PLD cleaves the phosphatidylcholine (lecithin) substrate to yield choline and phosphatidic acid. Second, choline is oxidized by choline oxidase to betaine and H2O2. Finally, H2O2, in the presence of horseradish peroxidase, reacts with the Amplex® Red reagent to generate the highly �uorescent product, resoru�n (excitation/emission maxima ~571/585 nm).

�e Amplex® Red Phospholipase D Assay Kit is designed for detecting PLD activity in cell ex-tracts and for screening PLD inhibitors.�is kit can be used to continuously assay PLD enzymes with near-neutral pH optima, whereas PLD enzymes with acidic pH optima can be assayed in a simple two-step procedure. Experiments with puri�ed PLD from Streptomyces chromofuscus indicate that the Amplex® Red Phospholipase D Assay Kit can detect PLD levels as low as 10 mU/mL using a reaction time of one hour (Figure 17.4.20). One unit of PLD is de�ned as the amount of enzyme that will liberate 1.0 micromole of choline from L-α-phosphatidylcholine per minute at pH 8.0 at 30°C. Each Amplex® Red Phospholipase D Assay Kit includes:

Figure 17.4.19 Detection of phosphatidylcholine-speci�c phospholipase C using the Amplex® Red Phosphatidylcholine-Speci�c Phospholipase C Assay Kit (A12218). Fluorescence was measured in a �uorescence mi-croplate reader using excitation at 560 ± 10 nm and �uores-cence detection at 590 ± 10 nm. The inset shows the sensitiv-ity at very low enzyme concentrations.

1200

1000

800

200

0

PC-PLC (mU/mL)

Fluo

resc

ence

600

12060 8040200

400

100

100

80

60

20

0

40

21 1.50.50

Figure 17.4.20 Quantitation of phospholipase D from Streptomyces chromofuscus using the Amplex® Red Phospholipase D Assay Kit (A12219). Fluorescence was mea-sured with a �uorescence microplate reader using excita-tion at 530 ± 12.5 nm and �uorescence detection at 590 ± 17.5 nm. The inset shows the sensitivity at very low enzyme concentrations (0–25 mU/mL).

2400

2000

1600

800

400

0

Phospholipase D (mU/mL)

Fluo

resc

ence

1200

300 4002001000

400

0201550 10

800

25

Figure 17.4.21 EnzChek® lipase substrate (E33955).

C OCH2

C OCH

CH2O(CH2)�CH3

(CH2)��

O��

N�

N

NH(CH2)��O

CO

NN(CH3)2N

H3C

• Amplex® Red reagent• Dimethylsulfoxide (DMSO)• Horseradish peroxidase (HRP)• H2O2 for use as a positive control

• Concentrated reaction bu�er• Choline oxidase from Alcaligenes sp.• L-α-Phosphatidylcholine (lecithin)• Detailed protocols







Each kit provides su�cient reagents for approximately 500 assays using a �uorescence microplate reader and a reaction volume of 200 µL per assay.

Fluorescent Substrates for Phospholipase D�e products of phospholipase A2, C and D cleavage of 1-O-alkyl-

2-decanoyl-sn-glycero-3-phosphocholine labeled with the BODIPY® FL �uorophore (D3771, Figure 17.4.10) can be separated and indepen-dently quantitated based on their di�erential migration on TLC or HPLC.23,47,48 Our BODIPY® FL analog is preferred for this application because it is relatively photostable and the �uorescence properties of its di�erent enzymatic products are all very similar.49 Researchers have taken advantage of these features to detect and quantitate phospholi-pase D activity in vascular smooth muscle cells,49,50 cultured mamma-lian cells 51 and yeast.52–54

EnzChek® Lipase Substrate�e triacylglycerol-based EnzChek® lipase substrate (E33955,

Figure 17.4.21) o�ers higher throughput and better sensitivity than chromogenic (TLC or HPLC) assays, and a visible light–excitable al-ternative to 6,8-di�uoro-4-methylumbelliferyl octanoate (DiFMU oc-tanoate, D12200; Section 10.6). In the presence of lipases, the non�uo-rescent EnzChek® lipase substrate produces a bright, green-�uorescent product (excitation/emission maxima of ~505/515 nm) for the accurate and sensitive detection of lipase activity in solution.

Figure 17.4.22 A) Phosphatidylcholines, phosphatidylinositols and phosphatidic acids are examples of glycerolipids derived from glycerol. B) Sphingomyelins, ceramides and cerebrosides are examples of sphingolipids derived from sphingosine. In all the structures shown, R represents the hydrocarbon tail portion of a fatty acid residue.

Ceramide

Sphingosine

HOCH2 CH CH

NH2 OH

CH CH(CH2)12CH3

Sphingomyelin

P

O

O

(CH3)3NCH2CH2O

C

R

O

+CH2 CH CH

NH OH

CH CH(CH2)12CH3O

CerebrosideOH

OH

HO

O

HO

O

R

C

O CH(CH2)12CH3CH

OHNH

CHCHCH2

C

R

O

HOCH2 CH CH

NH OH

CH CH(CH2)12CH3

Glycerol

CH

OH

HOCH2 CH2OH

Phosphatidylcholine

O CH2O

CH CH2O

+P

O

O

(CH3)3NCH2CH2O

O

R

C C

R

O

Phosphatidic Acid

O CH2O

CH CH2O

P

O

O

O

O

R

C C

R

O

Phosphatidylinositol

O CH2O

CH CH2O

P

O

O

O

R

C

O

OH

OH

HO

OH OH

C

R

O

A

B

Anti-Phosphoinositide Monoclonal AntibodiesPhosphatidylinositol (PI or PtdIns) and its phosphorylated deriva-

tives represent only a small fraction of eukaryotic cellular phospholip-ids but are functionally signi�cant in a disproportionately large number of regulatory and signal transduction processes.55–60 �e most famil-iar of these processes is the phospholipase C–mediated generation of the ubiquitous second messengers inositol 1,4,5-triphosphate (InsP3) and diacylglycerol (DAG) from phosphatidylinositol 4,5-diphosphate (PtdIns(4,5)P2; Section 17.2). Research has revealed the direct action of phosphatidylinositol 4,5-diphosphate (PtdIns(4,5)P2) and phospha-tidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P3) on a diverse array of cellular functions, including actin assembly and cytoskeletal dynam-ics, vesicular protein tra�cking, protein kinase localization and activa-tion, cell proliferation and apoptosis. We o�er mouse monoclonal IgM antibodies to PtdIns(4,5)P2 (A21327) and PtdIns(3,4,5)P3 (A21328) for immunocytochemical localization of these important lipid metabo-lites.61 Both antibodies have been shown to recognize their cognate phosphoinositides in murine and human cells with only slight cross-reactivity with other phosphoinositides or phospholipids.

SphingolipidsSphingolipids include sphingomyelins, which are phospholipid ana-

logs, as well as ceramides, glycosyl ceramides (cerebrosides), ganglio-sides and other derivatives (Figure 17.4.22). Several excellent reviews of the chemistry and biology of sphingolipids and glycosphingolipids and their role in the process of signal transduction are available.62,63








BODIPY® SphingolipidsCeramides (N-acylsphingosines), like diacylglycerols, are lipid second messengers that func-

tion in signal transduction processes.63–65 �e concentration-dependent spectral properties of BODIPY® FL C5-ceramide (D3521, B22650; Figure 17.4.23), BODIPY® FL C5-sphingomyelin 66–68 (D3522, Figure 17.4.24) and BODIPY® FL C12-sphingomyelin 69 (D7711) make them particularly suitable for investigating sphingolipid transport and metabolism,68,70–73 in addition to their appli-cations as structural markers for the Golgi complex 74 (Section 12.4). BODIPY® FL C5-ceramide can be visualized by �uorescence microscopy 75,76 (Figure 17.4.25, Figure 17.4.26) or by electron microscopy following diaminobenzidine (DAB) photoconversion to an electron-dense product.77 (Fluorescent Probes for Photoconversion of Diaminobenzidine Reagents—Note 14.2).

Our range of BODIPY® sphingolipids also includes the long-wavelength light–ex-citable BODIPY® TR ceramide 78,79 (D7540, Figure 17.4.27), as well as BODIPY® FL C5-lactosylceramide 80–85 (D13951), BODIPY® FL C5-ganglioside GM1

86 (B13950, Figure 17.4.28) and BODIPY® FL C12-galactocerebroside (D7519). All of our sphingolipids are prepared from D-erythro-sphingosine and therefore have the same stereochemical conformation as natural biologically active sphingolipids.87

Complexing �uorescent lipids with defatted bovine serum albumin (BSA) facilitates cell labeling by eliminating the need for organic solvents to dissolve the lipophilic probe; the

Figure 17.4.23 N-(4,4-di�uoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)sphingosine (BODIPY® FL C5-ceramide) (D3521).

Figure 17.4.24 N-(4,4-di�uoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)sphingosyl phosphocholine (BODIPY® FL C5-sphingomyelin) (D3522).

Figure 17.4.27 BODIPY® TR ceramide (N-((4-(4,4-di�uoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)phenoxy)acetyl)sphingosine) (D7540).

Figure 17.4.25 Nucleus and Golgi apparatus of a bovine pulmonary artery endothelial cell (BPAEC) labeled with Hoechst 33342 (H1399, H3569, H21492) and the BSA com-plex of BODIPY® FL C5-ceramide (B22650), respectively.

Figure 17.4.26 Cells in the notochord rudiment of a ze-bra�sh embryo undergoing mediolateral intercalation to lengthen the forming notochord. BODIPY® FL C5-ceramide (D3521) localizes in the interstitial �uid of the zebra�sh embryo and freely di�uses between cells, illuminating cell boundaries. This confocal image was obtained using a Bio-Rad® MRC-600 microscope. Image contributed by Mark Cooper, University of Washington.

Figure 17.4.29 Selective staining of the Golgi appara-tus using the green-�uorescent BODIPY® FL C5-ceramide (D3521) (top panel). At high concentrations, the BODIPY® FL �uorophore forms excimers that can be visualized using a red longpass optical �lter (bottom panel). The BODIPY® FL C5-ceramide accumulation in the trans-Golgi is su�cient for excimer formation (J Cell Biol (1991) 113:1267). Images con-tributed by Richard Pagano, Mayo Foundation.

Figure 17.4.28 BODIPY® FL C5-ganglioside GM1 (B13950).

Figure 17.4.30 Live J774 macrophage cells labeled with BODIPY® FL C5-ganglioside GM1 (B13950) and then with Alexa Fluor® 555 cholera toxin subunit B conjugate (C22843). Cells were then treated with anti–CT-B antibody to induce crosslinking. Yellow �uorescence indicates colo-calization of the two dyes. Nuclei were stained with the blue �uorescent Hoechst 33342 dye (H1399, H3570, H21492).







BSA-complexed probe can be directly dissolved in water.88 We o�er four BODIPY® sphingo-lipid–BSA complexes for the study of lipid metabolism and tra�cking, including:

• BODIPY® FL C5-ceramide (B22650)• BODIPY® TR ceramide (B34400)• BODIPY® FL C5-lactosylceramide (B34401)• BODIPY® FL C5-ganglioside GM1 (B34402)

BODIPY® FL C5-ceramide has been used to investigate the linkage of sphingolipid metabo-lism to protein secretory pathways 89–91 and neuronal growth.83,92 Internalization of BODIPY® FL C5-sphingomyelin (D3522) from the plasma membrane of human skin �broblasts results in a mixed population of labeled endosomes that can be distinguished based on the concentration-dependent green (~515 nm) or red (~620 nm) emission of the probe 68 (Figure 17.4.29). BODIPY® C5-sphingomyelin has also been used to assess sphingomyelinase gene transfer and expression in hematopoietic stem and progenitor cells.93 BODIPY® FL C5-lactosylceramide, BODIPY® FL C5-ganglioside GM1 and BODIPY® FL cerebrosides are useful tools for the study of glycosphingolipid transport and signaling pathways in cells.94–97 BODIPY® FL C5-ganglioside GM1 has been shown to form cholesterol-enhanced clusters in membrane complexes with amyloid β-protein in a model of Alzheimer disease amyloid �brils.98 Colocalization of �uorescent cholera toxin B conjugates (Section 7.7) and BODIPY® FL C5-ganglioside GM1 observed by �uorescence microscopy provides a direct indication of the association of these molecules in lipid ra�s 99,100 (Figure 17.4.30, Figure 17.4.31).

NBD SphingolipidsNBD C6-ceramide (N1154, Figure 17.4.32) and NBD C6-sphingomyelin (N3524) analogs

predate their BODIPY® counterparts and have been extensively used for following sphingolipid metabolism in cells 101–103 and in multicellular organisms.104 As with BODIPY® FL C5-ceramide, we also o�er NBD C6-ceramide complexed with defatted BSA (N22651) to facilitate cell loading without the use of organic solvents to dissolve the probe.88 Elimination of NBD �uorescence at the extracellular surface by dithionite reduction (Figure 17.4.33) can be used to assess endocyto-sis and recycling of NBD sphingolipids.

Amplex® Red Sphingomyelinase Assay Kit�e Amplex® Red Sphingomyelinase Assay Kit (A12220) is designed for measuring sphin-

gomyelinase activity in solution using a �uorescence microplate reader or �uorometer (Figure 17.4.34). �is assay should be useful for screening sphingomyelinase activators or inhibitors or for detecting sphingomyelinase activity in cell and tissue extracts. �e assay, which uses natural sphingomyelin as the principal substrate, employs an enzyme-coupled detection scheme in which phosphocholine liberated by the action of sphingomyelinase is cleaved by alkaline phosphatase to generate choline. Choline is, in turn, oxidized to betaine by choline oxidase, generating H2O2, which drives the conversion of the Amplex® Red reagent (A12222, A22177; Section 10.5) to red-�uorescent resoru�n (Figure 17.4.35). �is sensitive assay technique has been employed to detect activation of acid sphingomyelinase associated with ultraviolet radiation–induced apoptosis 105 and to characterize an insecticidal sphingomyelinase C produced by Bacillus cereus.106

Figure 17.4.31. A J774 mouse macrophage cell sequentially stained with BODIPY® FL ganglioside GM1 (B13950) and then with Alexa Fluor® 555 dye–labeled cholera toxin subunit B (C22843, C34776). The cell was then treated with an anti–CT-B antibody to induce crosslinking. Alexa Fluor® 555 dye �uorescence (left panel, red) and BODIPY® FL dye �uorescence (middle panel, green) were imaged separately and overlaid to emphasize the coincident staining (right panel, yellow). Nuclei were stained with blue-�uorescent Hoechst 33258 (H1398, H3569, H21491).

Figure 17.4.32 NBD C6-ceramide (6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)sphingosine) (N1154).

Figure 17.4.33 Dithionite reduction of 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid (NBD-X, N316). The elimination of �uorescence associated with this reaction, coupled with the fact that extraneously added dithionite is not membrane permeant, can be used to determine wheth-er the NBD �uorophore is located in the external or internal monolayer of lipid bilayer membranes.

N

O

N

NO2

NH(CH2)5 C

O

OH

N

O

N

NH2

NH(CH2)5 C

O

OH

S2O42−

Fluorescent Non�uorescent

Figure 17.4.34 Measurement of sphingomyelinase activ-ity using the Amplex® Red Sphingomyelinase Assay Kit (A12220). Each reaction contained 50 µM Amplex® Red re-agent, 1 U/mL horseradish peroxidase (HRP), 0.1 U/mL cho-line oxidase, 4 U/mL of alkaline phosphatase, 0.25 mM sphin-gomyelin and the indicated amount of Staphylococcus aureus sphingomyelinase in 1X reaction bu�er. Reactions were in-cubated at 37°C for 1 hour. Fluorescence was measured with a �uorescence microplate reader using excitation at 530 ± 12.5 nm and �uorescence detection at 590 ± 17.5 nm.

6,000

5,000

4,000

2,000

1,000

0

Sphingomyelinase (mU/mL)

Fluo

resc

ence

3,000

50300

800

400

00.60.40.20

1,000

10 20 40

600

200

Figure 17.4.35 Absorption and �uorescence emission spectra of resoru�n in pH 9.0 bu�er.








�e Amplex® Red Sphingomyelinase Assay Kit contains:

• Amplex® Red reagent• Dimethylsulfoxide (DMSO)• Horseradish peroxidase (HRP)• H2O2 for use as a positive control• Concentrated reaction bu�er• Choline oxidase from Alcaligenes sp.• Alkaline phosphatase from calf intestine• Sphingomyelin• Triton X-100• Sphingomyelinase from Bacillus sp.• Detailed protocols

Each kit provides su�cient reagents for approximately 500 assays using a �uorescence mi-croplate reader and a reaction volume of 200 µL per assay.

ADIFAB Reagent: A Unique Free Fatty Acid IndicatorElevated levels of free fatty acids (FFA)—which are associated with multiple pathological states,

including cancer, diabetes and cardiac ischemia 107—are generated by in�ammatory responses, phospholipase A activity and cytotoxic phenomena.108 Sensitive techniques are required to detect and quantitate free fatty acids because these important metabolites have low aqueous solubility and are usually found complexed to carriers. ADIFAB (A3880) is a dual-wavelength �uorescent FFA indicator that consists of a polarity-sensitive �uorescent probe (acrylodan, A433; Section 2.3) con-jugated to I-FABP, a rat intestinal fatty acid–binding protein with a low molecular weight (15,000 daltons) and a high binding a�nity for FFA 109–111 (Figure 17.4.17).

As shown in Figure 17.4.36, titration of the ADIFAB reagent with oleic acid results in a shi� of its �uorescence maximum from ~432 nm to ~505 nm. �e ratio (R) of these signals (505 nm/432 nm) can be converted to an FFA concentration by using the FFA dissociation constant (Kd) and employ-ing analysis procedures similar to those developed for Ca2+ indicators 112 (Chapter 19). Values of Kd vary considerably for di�erent fatty acids; a typical value is 0.28 µM for oleic acid 111 (determined at 37°C). �ere is little, if any, interference from bile acids, glycerides, sterols or bilirubin. With appropriate precautions, which are described in the product information sheet accompanying this product, ADIFAB can be used to determine FFA concentrations in the range 1 nM to >20 µM.

ADIFAB was used to investigate the physical basis of cis-unsaturated fatty acid inhibition of cytotoxic T cells.113 �is e�ect is due to inhibition of a speci�c tyrosine phosphorylation event that normally accompanies antigen stimulation.114,115 Measurements using ADIFAB have also revealed previously undetected di�erences in FFA binding a�nities among fatty acid–binding proteins from di�erent tissues 116,117 and have enabled quantitation of FFA levels in human serum as a potential diagnostic tool.107,118

Figure 17.4.36 The free fatty acid–dependent spectral shift of ADIFAB reagent (A3880). Spectra shown represent 0.2 µM ADIFAB in pH 8.0 bu�er with (+OA) and without (–OA) addition of 4.7 µM cis-9-octadecenoic (oleic) acid (OA). The ratio of �uorescence emission intensities at 505 nm and 432 nm can be quantitatively related to free fatty acid concentrations.

Flu

ores

cenc

e em

issi

on

_OA

+OA

400 450 500 550 600 650

Wavelength (nm)

Ex = 390 nm

1. Nat Rev Mol Cell Biol (2001) 2:327; 2. Curr Opin Struct Biol (2000) 10:737; 3. Physiol Rev (2000) 80:1291; 4. Biochim Biophys Acta (1994) 1212:26; 5. Biochim Biophys Acta (2000) 1488:124; 6. Mol Med Today (1999) 5:244; 7. FASEB J (1994) 8:916; 8. Cardiovasc Drugs �er (2009) 23:49; 9. Clin Chim Acta (2010) 411:190; 10. Methods Enzymol (2007) 434:15; 11. Circ Res (2009) 104:952; 12. Am J Physiol Gastrointest Liver Physiol (2009) 296:G445; 13. Science (2001) 292:1385; 14. Sci Signal (2009) 2:ra71-ra71; 15. J Invest Dermatol (2009) 129:2772; 16. Anal Biochem (1999) 276:27; 17. Science (2000) 288:1160; 18. Br J Pharmacol (1998) 124:1675; 19. J Biol Chem (1992) 267:21465; 20. J Biol Chem (2001) 276:12035; 21. J Lipid Res (2007) 48:385; 22. J Lipid Res (2007) 48:472; 23. Anal Biochem (1990) 185:80; 24. J Biol Chem (1999) 274:19338; 25. Anal Biochem (1981) 116:553; 26. Biochemistry (1995) 34:2049; 27. Biochim Biophys Acta (2000) 1486:321; 28. J Lipid Res (1992) 33:1863; 29. J Neurosci Methods (2000) 100:127; 30. Biochemistry (1999) 38:7803; 31. Biochemistry (1997) 36:14325; 32. Biochim Biophys Acta (1987) 917:411; 33. J Biol Chem (2001) 276:22732; 34. J Biol Chem (1999) 274:11494; 35. Biochemistry

(1999) 38:3867; 36. Anal Biochem (1995) 229:256; 37. Biochem J (1994) 298:23; 38. Proc Natl Acad Sci U S A (2004) 101:9745; 39. Mol Pharmacol (2000) 57:1142; 40. Proc SPIE-Int Soc Opt Eng (2000) 3926:166; 41. J Mol Biol (1998) 275:635; 42. Biochim Biophys Acta (1994) 1224:247; 43. J Biol Chem (1994) 269:4098; 44. J Biol Chem (1991) 266:1926; 45. J Neurochem (1991) 57:67; 46. J Biol Chem (2002) 277:45592; 47. Anal Biochem (2000) 286:277; 48. J Biol Chem (1997) 272:12909; 49. Anal Biochem (1994) 218:136; 50. J Biol Chem (1994) 269:23790; 51. Mol Biol Cell (1999) 10:3863; 52. Eukaryot Cell (2009) 8:1094; 53. Biochem J (1996) 314:15; 54. Biochem J (1995) 307:799; 55. Biochem J (2001) 360:513; 56. Biochem J (2001) 355:249; 57. Cell (2000) 100:603; 58. J Biol Chem (1999) 274:8347; 59. J Biol Chem (1999) 274:9907; 60. Annu Rev Cell Dev Biol (1998) 14:231; 61. J Histochem Cytochem (2002) 50:697; 62. Science (2010) 327:46; 63. Biochemistry (2001) 40:4893; 64. Trends Cell Biol (2000) 10:408; 65. J Biol Chem (1994) 269:3125; 66. Chem Phys Lipids (1999) 102:55; 67. Ann N Y Acad Sci (1998) 845:152; 68. Biophys J (1997) 72:37; 69. J Cell Biol (1998) 140:39; 70. Methods Enzymol (2000) 312:293; 71. Methods Enzymol (2000)

REFERENCES







DATA TABLE 17.4 PROBES FOR LIPID METABOLISM AND SIGNALINGCat. No. MW Storage Soluble Abs EC Em Solvent NotesA3880 ~15,350 FF,L,AA H2O 365 10,500 432 H2O 1A10070 880.68 FF,D,L DMSO 505 92,000 512 MeOH 2, 16A10072 986.67 FF,D,L DMSO 505 85,000 567 MeOH 2, 17, 18B3781 797.88 FF,D,L see Notes 342 75,000 471 EtOH 2, 3B3782 966.20 FF,D,L see Notes 340 62,000 473 EtOH 2, 4B7701 1029.80 FF,D,L see Notes 505 123,000 512 MeOH 2, 5B13950 1582.50 F,D,L DMSO, EtOH 505 80,000 512 MeOH 6B22650 ~66,000 F,D,L H2O 505 91,000 511 MeOH 6, 7B34400 ~66,000 F,D,L H2O 589 65,000 616 MeOH 7B34401 ~66,000 F,D,L H2O 505 80,000 512 MeOH 6, 7B34402 ~66,000 F,D,L H2O 505 80,000 511 MeOH 6, 7D3521 601.63 FF,D,L CHCl3, DMSO 505 91,000 511 MeOH 6D3522 766.75 FF,D,L see Notes 505 77,000 512 MeOH 2, 6D3771 854.86 FF,D,L see Notes 506 71,000 512 EtOH 2D3803 797.77 FF,D,L see Notes 503 80,000 512 MeOH 2, 8D7519 861.96 FF,D,L DMSO, EtOH 505 85,000 511 MeOH 6D7540 705.71 FF,D,L CHCl3, DMSO 589 65,000 616 MeOHD7711 864.94 FF,D,L DMSO 505 75,000 513 MeOH 6, 9D13951 925.91 FF,D,L DMSO, EtOH 505 80,000 511 MeOH 6D23739 1136.13 FF,D,L DMSO 505 92,000 511 MeOH 2, 10E33955 1011.15 F,D,L CHCl3 DMSO 505 94,000 515 MeOH 11H361 850.13 FF,D,L see Notes 342 37,000 376 MeOH 2, 12, 13H3809 856.09 FF,D,L see Notes 341 38,000 376 MeOH 2, 12, 13N1154 575.75 FF,D,L CHCl3, DMSO 466 22,000 536 MeOH 14N3524 740.88 FF,D,L see Notes 466 22,000 536 MeOH 2, 14N3786 771.89 FF,D,L see Notes 465 21,000 533 EtOH 2, 14, 15N3787 856.05 FF,D,L see Notes 465 22,000 534 EtOH 2, 14, 15N22651 ~66,000 F,D,L H2O 466 22,000 536 MeOH 7, 14For de�nitions of the contents of this data table, see “Using The Molecular Probes® Handbook” in the introductory pages.Notes

1. ADIFAB fatty acid indicator is a protein conjugate with a molecular weight of approximately 15,350. Em shifts from about 432 nm to 505 nm upon binding of fatty acids. (Mol Cell Biochem (1999) 192:87)

2. Chloroform is the most generally useful solvent for preparing stock solutions of phospholipids (including sphingomyelins). Glycerophosphocholines are usually freely soluble in ethanol. Most other glycerophospholipids (phosphoethanolamines, phosphatidic acids and phosphoglycerols) are less soluble in ethanol, but solutions up to 1–2 mg/mL should be obtainable, using sonica-tion to aid dispersion if necessary. Labeling of cells with �uorescent phospholipids can be enhanced by addition of cyclodextrins during incubation. (J Biol Chem (1999) 274:35359)

3. Phospholipase A cleavage generates a �uorescent fatty acid (P1903MP (Section 13.2)) and a �uorescent lysophospholipid.4. Phospholipase A cleavage generates a �uorescent fatty acid (P31 (Section 13.2)) and a �uorescent lysophospholipid.5. Phospholipase A cleavage results in increased �uorescence with essentially no wavelength shift. The cleavage products are D3862 (Section 13.2) and a �uorescent lysophospholipid.6. Em for BODIPY® FL sphingolipids shifts to ~620 nm when high concentrations of the probe (>5 mol %) are incorporated in lipid mixtures. (J Cell Biol (1991) 113:1267)7. This product is a lipid complexed with bovine serum albumin (BSA). Spectroscopic data are for the free lipid in MeOH.8. Phospholipase A2 cleavage generates a �uorescent fatty acid (D3834 (Section 13.2)) and a non�uorescent lysophospholipid.9. This product is supplied as a ready-made solution in the solvent indicated under "Soluble."10. Phospholipase A2 cleavage results in increased �uorescence with essentially no wavelength shift. The cleavage products are D3834 (Section 13.2) and a dinitrophenylated lysophospholipid.11. Fluorescence of the intact substrate is weak. Lipase hydrolysis releases a highly �uorescent fatty acid (D3823, Section 13.2).12. Pyrene derivatives exhibit structured spectra. The absorption maximum is usually about 340 nm with a subsidiary peak at about 325 nm. There are also strong absorption peaks below

300 nm. The emission maximum is usually about 376 nm with a subsidiary peak at 396 nm. Excimer emission at about 470 nm may be observed at high concentrations.13. Phospholipase A2 hydrolysis releases a �uorescent fatty acid; P31 (Section 13.2).14. Fluorescence of NBD and its derivatives in water is relatively weak. QY and τ increase and Em decreases in aprotic solvents and other nonpolar environments relative to water. (Biochemistry

(1977) 16:5150, Photochem Photobiol (1991) 54:361)15. Phospholipase A2 hydrolysis releases a �uorescent fatty acid; N316 (Section 13.2) from N3786 or N678 (Section 13.2) from N3787.16. Phospholipase A1 cleavage results in increased �uorescence with essentially no wavelength shift. The cleavage products are D3834 (Section 13.2) and a dinitrophenylated lysophospholipid.17. A10072 exhibits dual emission (Em = 510 nm and 567 nm in MeOH, 513 nm and 575 nm when incorporated in phospholipid bilayer membranes). Phospholipase A2 cleavage results in in-

creased 510–513 nm emission and reciprocally diminshed 567–575 nm emission.18. A10072 is also soluble at 2 mM in 2-methoxyethanol.

312:523; 72. Frontiers in Bioactive Lipids, Vanderhoek JV, Ed. 1996, p 203; 73. J Cell Biol (1996) 134:1031; 74. J Cell Biol (1991) 113:1267; 75. Cytometry (1993) 14:251; 76. J Cell Biol (1993) 120:399; 77. Eur J Cell Biol (1992) 58:214; 78. Mol Biochem Parasitol (2000) 106:21; 79. Infect Immun (2000) 68:5960; 80. J Cell Biol (2001) 154:535; 81. Am J Physiol Lung Cell Mol Physiol (2001) 280:L938; 82. Nat Cell Biol (1999) 1:386; 83. J Neurochem (1999) 73:1375; 84. Lancet (1999) 354:901; 85. Proc Natl Acad Sci U S A (1998) 95:6373; 86. J Cell Biol (1999) 147:447; 87. Biophys J (1999) 77:1498; 88. Cell Biology: A Laboratory Handbook, 2nd Ed., Vol. 2, Celis JE, Ed. 1998, p. 507; 89. Mol Biol Cell (1995) 6:135; 90. J Biol Chem (1993) 268:4577; 91. Biochemistry (1992) 31:3581; 92. J Biol Chem (1993) 268:14476; 93. Blood (1999) 93:80; 94. J Immunol (2007) 179:6770; 95. Mol Biol

Cell (2007) 18:2667; 96. J Cell Biol (2007) 176:895; 97. Methods (2005) 36:186; 98. J Biol Chem (2001) 276:24985; 99. Mol Biol Cell (2009) 20:3751; 100. J Cell Sci (2009) 122:289; 101. Biochim Biophys Acta (1992) 1113:277; 102. Adv Cell Mol Biol Membranes (1993) 1:199; 103. Biochim Biophys Acta (1991) 1082:113; 104. Parasitology (1992) 105:81; 105. J Biol Chem (2001) 276:11775; 106. Eur J Biochem (2004) 271:601; 107. Am J Cardiol (1996) 78:1350; 108. J Immunol (1991) 147:2809; 109. J Biol Chem (1995) 270:15076; 110. Biochemistry (1993) 32:7574; 111. J Biol Chem (1992) 267:23495; 112. Mol Cell Biochem (1999) 192:87; 113. Biochemistry (1993) 32:530; 114. J Biol Chem (1994) 269:9506; 115. J Biol Chem (1993) 268:17578; 116. Biochemistry (2000) 39:7197; 117. J Biol Chem (1994) 269:23918; 118. J Lipid Res (1995) 36:229.

REFERENCES—continued








PRODUCT LIST 17.4 PROBES FOR LIPID METABOLISM AND SIGNALINGCat. No. Product QuantityA3880 ADIFAB fatty acid indicator 200 µgA12218 Amplex® Red Phosphatidylcholine-Speci�c Phospholipase C Assay Kit *500 assays* 1 kitA12219 Amplex® Red Phospholipase D Assay Kit *500 assays* 1 kitA12220 Amplex® Red Sphingomyelinase Assay Kit *500 assays* 1 kitA21328 anti-phosphatidylinositol 3,4,5-triphosphate, mouse IgM, monoclonal RC6F8 (anti-PtdIns(3,4,5)P3) *1 mg/mL* 100 µLA21327 anti-phosphatidylinositol 4,5-diphosphate, mouse IgM, monoclonal 2C11 (anti-PtdIns(4,5)P2) *1 mg/mL* 100 µLB3781 1,2-bis-(1-pyrenebutanoyl)-sn-glycero-3-phosphocholine 1 mgB3782 1,2-bis-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine 1 mgB7701 1,2-bis-(4,4-di�uoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoyl)-sn-glycero-3-phosphocholine (bis-BODIPY® FL C11-PC) 100 µgB22650 BODIPY® FL C5-ceramide complexed to BSA 5 mgB13950 BODIPY® FL C5-ganglioside GM1 25 µgB34401 BODIPY® FL C5-ganglioside GM1 complexed to BSA 1 mgB34402 BODIPY® FL C5-lactosylceramide complexed to BSA 1 mgB34353 BODIPY® FL phosphatidylinositol(4,5) bisphosphate (BODIPY® FL PtdIns(4,5)P2) 50 µgD7540 BODIPY® TR ceramide (N-((4-(4,4-di�uoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)phenoxy)acetyl)sphingosine) 250 µgB34400 BODIPY® TR ceramide complexed to BSA 5 mgD3771 2-decanoyl-1-(O-(11-(4,4-di�uoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)undecyl)-sn-glycero-3-phosphocholine 1 mgD7519 N-(4,4-di�uoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)sphingosyl 1-β-D-galactopyranoside (BODIPY® FL C12-galactocerebroside) 25 µgD7711 N-(4,4-di�uoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)sphingosyl phosphocholine (BODIPY® FL C12-sphingomyelin) *1 mg/mL in DMSO* 250 µLD3803 2-(4,4-di�uoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (β-BODIPY® FL C5-HPC) 100 µgD3521 N-(4,4-di�uoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)sphingosine (BODIPY® FL C5-ceramide) 250 µgD13951 N-(4,4-di�uoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)sphingosyl 1-β-D-lactoside (BODIPY® FL C5-lactosylceramide) 25 µgD3522 N-(4,4-di�uoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)sphingosyl phosphocholine (BODIPY® FL C5-sphingomyelin) 250 µgD23739 N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-2-(4,4-di�uoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-

phosphoethanolamine, triethylammonium salt (PED6)1 mg

E10215 EnzChek® Direct Phospholipase C Assay Kit *phosphatidylcholine speci�c* *2-plate size* 1 kitE10216 EnzChek® Direct Phospholipase C Assay Kit *phosphatidylcholine speci�c* *10-plate size* 1 kitE33955 EnzChek® lipase substrate *green �uorescent, 505/515* 100 µgE10219 EnzChek® Phospholipase A1 Assay Kit *2-plate size* 1 kitE10221 EnzChek® Phospholipase A1 Assay Kit *10-plate size* 1 kitE10217 EnzChek® Phospholipase A2 Assay Kit *2-plate size* 1 kitE10218 EnzChek® Phospholipase A2 Assay Kit *10-plate size* 1 kitH361 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (β-py-C10-HPC) 1 mgH3809 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphoglycerol, ammonium salt (β-py-C10-PG) 1 mgN1154 NBD C6-ceramide (6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)sphingosine) 1 mgN22651 NBD C6-ceramide complexed to BSA 5 mgN3524 NBD C6-sphingomyelin (6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)sphingosyl phosphocholine) 1 mgN3787 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD C12-HPC) 5 mgN3786 2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD C6-HPC) 5 mgA10070 PED-A1 (N-((6-(2,4-DNP)amino)hexanoyl)-1-(BODIPY® FL C5)-2-hexyl-sn-glycero-3-phosphoethanolamine) *phospholipase A1 selective substrate* 100 µgP6466 phospholipase C, phosphatidylinositol-speci�c *from Bacillus cereus* *100 U/mL* 50 µLA10072 Red/Green BODIPY® PC-A2 (1-O-(6-BODIPY® 558/568-aminohexyl)-2-BODIPY® FL C5-sn-glycero-3-phosphocholine) *ratiometric phospholipase A2 substrate* 100 µg



Documents

CHAPTER 1 CHAPTER 17 Fluorophores and Probes for Their ... · Molecular Probes ™ Handbook A Guide to Fluorescent Probes and Labeling Technologies ... BioProbes® Journal of Cell