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| Life Technologies Proprietary & Confidential | 2
Direct VS. Secondary Detection
Direct-Labeled Primary Antibody
• Low background
• Covalent attachment of label via amine-reactive dyes to lysines
• Labeling could affect antigen recognition site
• Fluorescent signal similar to Zenon®-Labeled Antibody
Indirect-Labeled Secondary Antibody or Streptavidin
• Higher Background
• Brighter Signal
Zenon®-Labeled Antibody
• Low background
• Non-covalent attachment of Zenon® fragment to Fc portion
• Labeling will NOT affect antigen recognition site
• Fluorescent signal similar to Direct-labeled Primary antibody
• For use with “higher” abundance targets
• To reduce background due to non-specific secondary detection
• For use with multiplexed analyses with same-species antibodies
| Life Technologies Proprietary & Confidential | 3
How to Choose an Antibody Labeling Kit....1) Do I need a directly labeled conjugate?
2) What
label?
For an interactive version visit:
www.invitrogen.com/spectraviewer
| Life Technologies Proprietary & Confidential | 4
How to Choose An Antibody Labeling Kit....3) How much antibody do I want or need to label?
1-20 µµµµg IgG 20-100 µµµµg IgG 100 µµµµg IgG 1 mg IgG
Zenon® IgG Labeling Kits
APEX Antibody Labeling Kits
Microscale protein labeling kits
Monoclonal antibody labeling kits
“Original” Protein labeling Kits
| Life Technologies Proprietary & Confidential | 5
How to Choose An Antibody Labeling Kit....4) Should the label be covalently or non-covalently
attached to the antibody?Covalent attachment Non-Covalent label attachment
These conjugates are stable for months to even years…
Can be used with any antibody application
Products:
• APEX Antibody Labeling kits
• Microscale protein labeling kits
• Monoclonal antibody labeling kits
• Protein labeling kits
Conjugates are stable for hours not days…
Recommended for use with applications requiring short incubations
NOT recommended for immunohistochemistry (IHC) applications requiring several hours to overnight antibody incubations
Product:
• Zenon® Antibody Labeling Kits
Label directly & covalently attached to the IgG antibody
Label non-covalently attached to the IgG antibody using directly labeled anti-FC fab fragments
| Life Technologies Proprietary & Confidential | 6
How to Choose an Antibody Labeling Kit5) Does the antibody contain stabilizing proteins?Typically:
• The stabilizing is bovine serum albumin (BSA)
• This is used to stabilize small amounts of packaged IgG antibodies
• Whether the IgG contains BSA will either be listed on the vial or in the product data sheet
Why does this matter?
If labeling with a microscale, monoclonal & protein labeling kit, these kits utilize amine-reactive dyes that will react with the stabilizing proteins in addition to the IgG antibody. If using a kit with amine-reactive dyes and the stabilizing proteins are not removed, the antibody will be underlabeled and produce a very weak signal.
Zenon® and APEX antibody labeling kits are completely compatible with stabilizing proteins.
| Life Technologies Proprietary & Confidential | 7
Zenon® vs non-Zenon® Antibody Labeling KitsZenon® Antibody Labeling
APEX, Microscale, Monoclonal Antibody & Protein Labeling Kits
• Fastest way to label IgG (10 minutes!!)
• Very, very easy-to-use
• Perfect for small quantities of IgG (<20 µµµµg)
• Great way to attach, Alexa Fluor® dyes R-PE, R-PE-tandems (and other fluorescent proteins or enzymes)
• Fast (1.5-2 hours, only 15-30 minutes of actual hands-on time)
• Very easy-to-use
• Perfect for larger quantities of IgG (>20 µµµµg – 1 mg)
• Great way to covalently attach Alexa Fluor® dyes & biotin
| Life Technologies Proprietary & Confidential | 8
~ 1 µg primary antibody
Add Zenon®
reagent at desired molar ratio
Incubate 5 minutes
Block with non-specific IgG
& use directly
How does Zenon Labeling work?
| Life Technologies Proprietary & Confidential | 9
Kinetically trapped complex formation: key to making the system work.
Fast complex formation Slow complex dissociation
+ Alexa Fluor 488 ZenonCapture by BSA-biotin in microtiteer plate
Mouse anti-biotin IgG1
Control IgG1
Blocked mouse anti-biotin IgG1
+ Alexa Fluor 488 ZenonCapture by BSA-biotin in microtiteer plate
Zenon labeling technology- Stability
| Life Technologies Proprietary & Confidential | 10
Use in imaging…
• anti-nuclear mouse mAb + Alexa Fluor 350 Zenon labeling reagent• F-actin + Alexa Fluor 488 phalloidin• anti-tubulin mouse mAb + Alexa Fluor 568 Zenon labeling reagent
| Life Technologies Proprietary & Confidential | 11
Use of Zenon® Labeling Technology in flow cytometry
• Human peripheral blood lymphocytes were stained with the following three antibodies: an anti-CD3 mouse IgG1 antibody (A21330) prelabeled with the Zenon® Alexa Fluor® 647 Mouse IgG1 Labeling Kit (Z25008), an anti-CD4 mouse IgG1 antibody (A21334) prelabeled with the Zenon® R-Phycoerythrin Mouse IgG1 Labeling Kit (Z25055) and an anti-CD8 mouse IgG2a antibody (A21338) prelabeled with the Zenon® Alexa Fluor® 488 Mouse IgG2a Labeling Kit (Z25102). Panels A and B show that cells can be separated by plotting the orange-fluorescent versus green-fluorescent signal or red-fluorescent versus orange-fluorescent signal, respectively, demonstrating that Zenon® labeling technology does not transfer to other antibodies in the same sample. The samples were analyzed on a Coulter Elite flow cytometer using 488 nm excitation for R-phycoerythrin and the Alexa Fluor® 488 dye, and 633 nm excitation for the Alexa Fluor® 647 dye.
| Life Technologies Proprietary & Confidential | 12
APEX Antibody Labeling Kits• Convenient: APEX Kits label small amounts of antibody, 10-20 µµµµg
• Covalent attachment of label: Once attached, label will not come off
• Compatible: Stabilizing proteins (i.e., BSA) or amine-containin g buffers (i.e., TRIS) will not interfere with labeling
Customer requirements:
• 10-20 µg IgG antibody
• Standard pipette (for 200 µL volume)
• ~15 minutes “hard labor” (2.5 hours total)
With pipette, apply to APEX labeling tip
• Apply IgG antibody
• Fluorescent label
• Wash buffers
APEX antibody labeling tip
With pipette, elute
• Wash buffers
• Labeled IgGMultiple steps: Customer will need to either apply or elute
| Life Technologies Proprietary & Confidential | 13
APEX or Zenon ?
APEX Labeled Antibody
• Lowest background
• Labeling could affect antigen recognition site
• Fluorescent signal similar to Zenon®-Labeled Antibody
• 10-20 µµµµg IgG per labeling
• Attach simple, organic dyes (Alexa Fluor®, Oregon Green® dyes for use in flow cytometry or microscopy or even as haptens with anti-Alexa Fluor or Anti-Oregon Green antibodies)
Indirect-Labeled Secondary Antibody or Streptavidin
• Higher Background
• Brightest Signal
Zenon®-Labeled Antibody
• Low background
• Fc-attachment of label. Labeling will NOT affect antigen recognition site
• 1-20 µµµµg IgG per labeling
• Isotype specific labeling (IgG2a or IgG2b
• Attach
• Simple organic dyes (for use in flow cytometry or microscopy)
• Phycobiliproteins (R-PE, APC for flow cytometry)
• Tandems (Alexa Fluor-APC for flow cytometry)
Antibody
Antigen
Label
| Life Technologies Proprietary & Confidential | 14
Antibody & Protein Labeling Kits OverviewAPEX Antibody Labeling Kits
Microscale Protein Labeling Kits
Monoclonal Antibody Labeling Kits
Protein Labeling Kits
*Requires optimized primary antibodies to target or short incubations (<1 hour)
Flow Cytometry
Microscopy
• ICC
• IHC
Applications
Zenon® Antibody Labeling Kits
SAIVI™ Protein / Antibody Labeling Kits
QDot® Nanocrystal Secondary Antibody Labeling Kits
Applications
Small Animal in-vivoImaging (SAIVI™) Applications
Flow Cytometry
Microscopy
• ICC
• IHC*
Applications*
Flow Cytometry *
Microscopy *
• ICC
• IHC
Western blots
SAIVI™ Applications
Applications
* Verify ex/em resources
Available Labels
Alexa Fluor® Dye Series
Biotin and DSB™-X biotin
Oregon Green® Dyes
Pacific™ Dyes
Standard Dyes (fluorescein)
Available Labels
Near IR Alexa Fluor® Dyes
• Alexa Fluor® 647, 680 & 750
Available Labels
Alexa Fluor® Dye Series
Alexa Fluor® tandems
Phycobiliproteins (R-PE, APC)
Biotin and DSB™-X biotin
Oregon Green® Dyes
Pacific™ Dyes
Standard Dyes (fluorescein)
Available Labels
QDot® Nanocrystals
• 525, 565, 585, 605, 655, 705, 800
• Covalent label attachment
• DOL optimized for brightest signal
• Covalent label attachment
• DOL optimized for SAIVI™Applications
• DOL control reagent
• 5 minute antibody labeling
• Non-covalent label attachment
• Organic dyes & R-PE
• Covalent label attachment
Jean-Michel COCCHI – TSSAnalyse de la [email protected]
Fluorescence amplification with Tyramide kits
| Life Technologies Proprietary & Confidential | 16
TSA™ can provide high resolution and high S/N
Tyramide Signal Amplification (TSA™)
| Life Technologies Proprietary & Confidential | 17
Why Use TSA?
• Generates multiple copies of Alexa Fluor dyes at ta rget site
• Gives high-resolution signal amplification
• When normal amplification methods fail− Low abundance targets
Calbindin Revealed
| Life Technologies Proprietary & Confidential | 18
StreptavidinAlexa Fluor
546
Alexa Fluor 546 TSA
Alexa Fluor 546 Dye FISH Signal: Streptavidin vs TSA
0
500
1000
1500
RFU
AF546-SA AF546-TSA
10-fold increase in sensitivity w/ TSA™
Chromosome FISH: Labeled Streptavidin vs TSA™
| Life Technologies Proprietary & Confidential | 19
Labelling examples on Zebrafish sample
A zebrafish retina cryosection labeled with the mouse monoclonal antibody FRet 43 and detected using TSA Kit #9 with the HRP conjugate of goatanti–mouse IgG antibody and Alexa Fluor® 488 tyramide.
| Life Technologies Proprietary & Confidential | 20
Labelling examples on Zebrafish sample
Zebrafish larva reticulospinalneurons. AlexaFluor® 488 tyramide .
| Life Technologies Proprietary & Confidential | 21
Why Use TSA?• Increased sensitivity
− ~100-fold compared to avidin–biotinylated enzyme complex (ABC)
• Generates multiple copies of AlexaFluor dyes at target site
• Gives high-resolution signal amplification
• When normal amplification methods fail
− Low abundance targets
3 color sequential TSA™
| Life Technologies Proprietary & Confidential | 22
Protecting the fluorescence signal-Antifade Reagents
Prolong® Gold
•Optimized formulation-Superior antifade with little or no impact on initial fluorescence intensity
•Ready to use - dropper bottle
•Cures to a firm gel – no more nail polish
NEW: SlowFade® Gold - antifade mounting media which doesn’t polymerize