473

Gary B. Willars and R.A. John Challiss (eds.), Receptor Signal Transduction Protocols: Third Edition, Methods in Molecular Biology, vol. 746, DOI 10.1007/978-1-61779-126-0, © Springer Science+Business Media, LLC 2011

Index

A

Abbe’s law ...................................................................... 426Acceptor ...................................54, 299, 300, 305, 311, 312,

314, 358–363, 367–369, 374, 375, 377, 380–385Acceptor-ligand .............................. 375, 379, 380, 382–385Adenovirus ...............................................39–42, 46, 49, 50Adenylyl cyclase ............................................................ 298a2-Adrenergic receptor .................................................. 349Affinity .............................7, 54–56, 95, 100, 135–137, 147,

157, 158, 162, 163, 166, 196–202, 204, 205, 207, 208, 211–213, 215, 217, 221, 232, 239, 247, 274, 283, 285, 286, 302, 303, 330, 331, 354, 376, 390, 391, 393–395, 399–408, 411, 412, 415, 428, 438

Affinity cooperativity ......................................197, 202, 207Agarose gel .............................44–46, 60, 69, 71, 72, 74–75,

82, 83, 90, 92, 93, 96, 176Agarose gel electrophoresis ............................62, 64, 74–77,

92, 174, 176Agar plates ......................................................63, 73, 92, 94Agonist-directed trafficking .......................................... 268A-kinase anchoring proteins (AKAPs) ..................298, 302,

304, 347Alanine scan .........................................................86, 87, 95Allosteric ..................................................85, 195–208, 298Allosteric agonism ......................................................... 204Alternative splicing ........................................................ 169Amplicon ........................................ 167, 179, 180, 182, 184Amplification .................................... 10, 47, 80, 91–93, 95,

96, 139, 165–168, 171, 177, 183, 184, 258, 268, 291, 318, 403

Analysis of covariance (ANCOVA) ............................... 468Annealing .................................... 33, 59, 66–68, 76, 80, 95,

167, 168, 178–180, 183Antibiotic ...................................8–9, 11, 12, 14, 19, 23, 34,

36, 45, 63, 65, 70, 71, 92, 94, 102, 103, 107, 111, 254, 337, 349, 351, 377, 401, 420

Antibiotic resistance .......................................12, 45, 63, 92Antibody(ies) ..............................31, 53, 102, 118, 166, 238,

252, 264, 298, 323, 375, 392, 400, 415, 426, 452 Antibody production ............................................. 246–247A priori tests .......................................................... 451–454Arrestin ............. 86, 100, 330, 358, 359, 361, 363–367, 369

Association constant .............................................. 154, 363Autocorrelation............................... 226–229, 232, 234, 235Autofluorescence .....219, 222, 230, 234, 235, 288, 313, 340Autoradiogram .............................................................. 242Autoradiography ..............135, 243, 264, 350, 351, 353, 418

B

Background subtraction ................................................. 294Bait...... ........................................... 390, 392, 394–397, 400Bartlett’s test .................................................................. 451b-galactosidase ...........................................23, 33, 126–130Biocytin maleimide .......................................................... 87Bioluminescent resonance energy transfer

(BRET) ..............................................357–370, 374Biosensors ............................... 105, 106, 297–315, 329–342Biotin.. .......................54, 117, 400, 405, 428, 430, 436, 440Biotinylation ........................... 428, 430–431, 435–438, 440BiP......... ............................................................................427Bit depth.................................................281, 285, 290–292BLAST.............................................................91, 171, 180Blasticidin ....................................... 9, 12, 23, 24, 27, 29–33Bleaching ..................................57, 229, 235, 285, 287, 288,

293, 310, 313, 314Bleed-through ................................ 220, 312, 340, 383, 438Blunt cloning ............................................................. 90, 94Bmax...... ............................. 136, 144, 145, 159, 428, 466, 468Bonferroni ......................................................455, 456, 463BRET2 ............................................358–360, 362–365, 369BRET3 .................................... 358–360, 362, 363, 368, 369BRET ratio ....................................................363, 366, 369Butyrate ........................................................................... 19

C

Ca2+ indicator ..........279–281, 283–287, 289, 290, 293, 294Ca2+ ionophore....................................................... 279, 280Calcium ............. 55, 117, 196, 277–295, 376, 378, 379, 400Calcium phosphate .............................................8, 413, 416Calf intestinal alkaline phosphatase

(CIAP) ...............................................240, 247, 248Calibration of fluorescence .................................... 285, 294Calmodulin affinity chromatography .................... 404–406Calnexin ........................................................................ 427

474 ReceptoR Signal tRanSduction pRotocolS

Index

Cameleons ..................................................................... 278Camgaroos..................................................................... 278Ca2+ puff ................................................................ 282, 283Cardiomyocytes .......307, 308, 318, 319, 321–323, 325, 327Ca2+-sensitive dye ........................... 288, 329, 333, 338, 340Caveolae ................................................................ 411, 412Caveolin ......................................... 412, 415, 417–419, 421cDNA.. ...............................3, 4, 6–8, 10, 21, 31, 32, 41–43,

45, 46, 53, 62–69, 71, 76, 79, 102–105, 107, 108, 110, 139, 165, 175–180, 185–188, 302, 319, 333, 337, 348–350, 359, 361, 365, 367

Cell fixation ..................................................... 57, 429–434Cell lysate .......................................... 31, 35, 124, 125, 140,

256, 299, 324, 347, 396Cesium purification ......................................................... 47Chemiluminescence........................................164, 255, 437Cheng–Prusoff ...............................................147, 148, 161Chimeric mutagenesis ....................................89–90, 92–94Chi square test ............................................................... 461Cholera toxin B (CTB) ..........................414, 417, 418, 420Cholesterol ............................. 411, 412, 415, 419, 421, 422Chromosomal integration................................................ 18Clonal isolation ......................................................... 12–14Cloning rings ............................................................. 32, 36CMV... ..................................................5, 7, 33, 43, 256, 402Codon.. ...................................32, 34, 55, 62, 64, 66–69, 73,

79, 81, 82, 90, 123, 361Coelenterazine ................................ 357, 358, 360–366, 368Competence ............................................................ 96, 121Competent cells .................43–44, 63, 68, 80, 83, 89–92, 94Competition binding ..............................198, 211, 224–225Competitive antagonist ......................................... 196, 200Concanavalin A ............................................................. 427Confidence interval (CI) ........................202, 205, 447, 467Confocal fluorescence microscopy ......................... 426, 434Confocal microscopy .............................105–107, 122, 212,

218, 283, 339, 361, 439Coomassie ......................................................129, 245, 321Cooperativity factor ................................197, 198, 204, 205Co-transfection .................................. 29, 34, 104, 105, 302Cq........ .................................................................. 184–186Cryopreservation ............................................................. 34Curve fitting ................................... 198, 234, 364, 466–470CXCR4 ..................................................252–254, 256–258Cyan fluorescent protein (CFP) ......................25, 300–303,

305, 306, 309–314, 330, 374Cyclic 3′,5′-adenosine monophosphate

(cAMP) ...................... 214, 297–305, 309, 311–314

D

Data transformation ....................... 145, 445, 446, 460, 466DeepBlueC ............................................................ 358, 360De-esterification ............................................................ 280Degrees of freedom ........................ 148, 450, 452, 463–465

Desensitization ................................. 58, 304, 359, 390, 399Detergent extraction .............................................. 411, 419Detergent-insoluble glycolipid-enriched

membranes......................................................... 412Detergent-resistant-membranes (DRMs) ............. 411–422Diacylglycerol (DAG) .................... 318, 330, 336, 338, 342Diagnostic digestion ............................................ 70–72, 75Dialysis ...........................................................321, 326, 352Dichroic mirror .............................................220, 228, 278,

287, 306, 309, 313Diffusion time ....................................................... 228, 229Dimerisation .................................................................... 31Dispersion ......................................................444, 446–448Dissociation constant ............................ 136, 137, 145, 197,

198, 200, 201, 203DNA concentration ........................................64, 71, 72, 75DNA extraction ............................................................... 75DNA polymerase .........60, 67, 76, 80, 89, 91, 166–168, 177DNase. ................................................78, 82, 174–176, 178Dominant negative ................. 100–102, 105, 107, 110, 257Donor.. .....................................299, 300, 305, 311, 312, 314,

358–361, 363, 367–369, 374–376, 380–385Donor-ligand .................................. 375, 379, 380, 382–385Down-regulation ........................................................... 428Doxycycline .......................5, 7, 9, 15, 22, 24–27, 29, 31, 35Dropout mix .................................................................. 116Dual affinity tag ............................................................ 402Duncan’s multiple range test ......................................... 456Dunnett’s test ........................................................ 456, 468Dunn’s test ............................................................. 461, 462Dwell time ..............................................226, 227, 230, 235Dynamin ....................................................................... 257

E

Ecdysone .................................................. 6, 7, 9, 10, 15, 18Edman degradation ........................................238, 243–245EEA1 .............................................................................427Efficacy ...........................5, 86, 95, 196, 197, 204, 205, 207,

208, 211–215, 217, 221, 232, 264, 282, 408Efficacy cooperativity .............................197, 204, 205, 208Electrophoresis ............................ 31, 62, 64, 65, 71, 74–77,

82, 92, 118, 174–176, 241, 242, 324, 347, 350, 390, 392, 394, 397, 405, 408, 414, 437

Electroporation ................................ 8, 43, 44, 49, 104, 111, 121–122, 323, 376–378, 384

ELISA. .............................................................58, 247, 428Emission spectra ............................ 213, 216–221, 286, 312,

338, 358, 365, 368Endocytosis .................................... 257, 412, 427, 432, 435Endonuclease............................ 10, 60, 75–76, 78, 168, 169Endosomal sorting complex required for transport

(ESCRT) ........................................................... 252Endosome.......................................................139, 251, 427EnduRen ................................................358, 360, 362, 368

ReceptoR Signal tRanSduction pRotocolS

475

Index

Enhanced GFP (eGFP) .......................... 57, 60, 62, 68, 69, 71, 72, 105–108, 110, 111, 331–334, 336, 339, 340, 342, 358, 360, 363

Epac.... ............................................................300–305, 308Epifluorescence.......................................305, 426, 434, 439Epitope tag .................................... 24, 31, 53–83, 118, 126,

239, 248, 257, 426, 438Error.... .................................... 160, 271, 443, 446, 449–451,

453–459, 462–465Ethidium bromide ..............................................74, 83, 190Europium .............................................................. 374, 375Excitation spectra .................................................. 220, 299Experimental design ............... 188, 443, 444, 450, 456, 465Experimental power ...................................................... 463Expression ..........................3–19, 21–36, 39–50, 53, 86, 99,

114, 165, 195, 237, 253, 266, 304, 326, 331, 349, 359, 374, 400

Extended BRET (eBRET) .................................. 358–360, 362, 363, 368, 369

F

Fisher’s exact test ........................................................... 461Fixation ....................................................57, 426, 429–435FLAG-tag ..........................................................26, 55, 253Flotation ................................................................ 411, 415Flotillin–1 ...............................................415, 417–419, 421Flow cytometry....................... 287, 427–428, 430, 434–435Flp-In™ ...................................................................... 21–36Flp recombination target (FRT) ............................... 22–25,

28–30, 33–35, 66Fluo–3 .................................................... 282, 283, 288, 293Fluorescence

correlation spectroscopy (FCS) ........212, 213, 218–219, 226–232, 234, 235

life-time ....................................................213, 305, 374microscopy ................................. 57, 108, 111, 122–123,

212, 403, 406, 426, 427polarisation ...................................................... 212, 215

Fluorescent biosensor ............................................ 329–342Fluorescent ligand ..................................211–235, 373–386Fluorophore ....................166, 168, 169, 212–220, 226–230,

232–235, 299, 300, 302, 305, 308, 309, 312, 313, 330, 338, 341, 358, 361, 374, 375, 383, 433, 435

Fluoview ................................................................ 332, 340Focal plane ..............................................222, 313, 314, 434Focus drift ............................................................. 310, 314Förster radius ................................................................. 299Förster resonance energy transfer (FRET) ............168, 213,

297–315, 330, 373–386FRET ratio .....................................................306, 315, 383F-test... .................................................................. 147, 148Fura–2............................................. 284, 286–288, 290, 338Fura-PE3 ....................................................................... 290Fura red ..................................................288, 332, 333, 338

G

G12....... ......................................................................317–327G418... .................................................. 9, 12, 27, 28, 32, 36Galactosyl transferase .................................................... 427Ganglioside GM1 ......................................................... 415Ga-protein antisera ....................................................... 272Gaussian distribution...................... 445, 446, 448, 460, 465GC clamp .............................................................81, 91, 95GDP ................................113, 114, 263–268, 270–273, 317GelGreen ....................................................................... 190Gel overlay............................................................. 347–354GelRed™ ..........................................60, 72, 74, 83, 176, 190GeneSwitch ........................................ 6, 7, 9, 10, 15, 17, 18Geneticin ....................................................27, 34, 401, 403Genomic DNA (gDNA) ........................167, 173, 174, 180Glutathione sepharose ............................319, 320, 324–327Glutathione-S-transferase (GST) ...........54, 247, 317–321,

323–326, 347–354, 390Glycerol stock ............................................................ 63, 71Glycolipids .................................................................... 411Gpa1.... .................................................................. 115, 128GPCR-associated protein complexes ................... 389–397,

399–408G protein ................................22, 86, 88, 95, 100, 113, 114,

237, 263–274, 298, 317–327, 330, 359, 389, 392, 396, 401, 406

G protein-coupled receptor kinase (GRK) ........... 100–103, 105–110, 396

Gradient centrifugation ................................................. 125 GraphPad Prism™ .......................... 197, 201, 203, 206, 232,

335, 449, 453, 460, 462, 468, 470Green fluorescent protein (GFP) ................................. 26, 32, 43,

46–50, 54, 55, 57, 58, 122, 126, 213, 234, 302, 323, 330, 338, 359, 374, 426, 439

GSH-agarose ..........................................348, 349, 352–354GST-fusion protein ........................................317, 348–354GTP..... ............ 113, 114, 201, 263, 264, 268, 273, 317, 323GTPase ........................................... 264, 302, 317, 318, 323GTPase-activating proteins ........................................... 114[35S]GTPgS ............................................263–274, 317, 318GTPgS-binding ..............................................263–274, 318

H

HA-tag ..................................56, 66, 68, 69, 79, 81, 82, 253Helmert contrast ........................................................... 453Heterodimer .......................................................7, 374, 375Heterologous expression .................................. 99, 113–130Hill slope ................................................146, 147, 150, 161His-tag ...............................................................55, 56, 396[3H]-N-methyl scopolamine (NMS) ......197, 200–203, 208Homodimer ............................................124, 129, 374, 375HPLC.. .....................................................................216–219hRluc... ..............................................................................368

476 ReceptoR Signal tRanSduction pRotocolS

Index

Hydrophilic ligands ....................................................... 139Hygromycin ...................9, 11, 12, 19, 24, 27, 28, 30–32, 34

I

IC50.... ......................................................................146, 147ICUE... .................................................................. 301, 303Imaging ....................................57, 105, 110, 213, 216, 217,

221–226, 230–234, 277–295, 300–308, 312–314, 329, 331–335, 337–341, 358, 360, 362, 412, 418, 426, 433, 434, 438, 439

Immobilized metal affinity chromatography (IMAC) ..... 56Immunisation ................................................................ 247Immunoblotting ................56, 118–119, 129, 252, 253, 257Immunocytochemistry (ICC) .............................57, 58, 239Immunoelectron microscopy ......................................... 427Immunoprecipitate ................. 238, 248, 256, 258, 264, 273Immunoprecipitation ............................... 56, 247, 253, 256,

265, 267, 271–273, 374, 428, 436Immunoreactivity ............................. 55, 427, 428, 435, 437Indo–1. ......................................................................287–288Inducible expression .........................................3–19, 32, 95Inducible locus ........................................................... 31, 32Induction ............. 5, 6, 9, 10, 14, 15, 19, 22, 24–26, 35, 282Infection .................................................................... 39–50Infinite dilution ............................................................. 138Inhibition ..........................................................7, 106, 107,

136, 137, 139, 140, 146–151, 159–162, 177, 201, 203, 214, 302

Inositol 1,4,5-trisphosphate............................277, 318, 330Intercalating dye ..................... 166, 167, 169, 171, 183, 191Internalization ....................................... 139, 251, 252, 257,

359, 374, 380, 427, 428Iodoacetamide ................................................246, 256, 431IPTG... .................. 6, 7, 10, 15, 94, 318, 319, 326, 349, 351Isosbestic point .............................................................. 287ITRAQ .......................................................................... 239

K

K+1....... ............................................................136, 137, 159K–1........ ..................................................136–138, 152, 153,

155, 159, 162, 451Kd........ ............................................................................ 95Ki......... .................................................................. 148, 376Kill curve ................................................................... 34, 36Kolmogorov–Smirnov test ............................................. 448Kozak.. ................................................ 10, 32, 64, 67, 68, 79Kruskal–Wallis test ................................................ 461, 462

L

LacSwitch II ...................................................6, 7, 9, 10, 15LAMP. .................................... 287, 306, 308, 312, 332, 427Lanthanides ........................................................... 374, 377Least significant difference test ..................................... 455Levene test ............................................................ 448, 451

Ligand binding .................................... 7, 10, 14, 16, 54, 58, 86, 87, 95, 115, 135–164, 195–208, 217, 221–225, 231, 232, 264, 265, 361, 376, 399, 403, 406, 407, 428, 445, 466

Ligase.. ..................33, 44, 59, 65, 68, 78, 79, 90, 93, 94, 252Ligation ............................................. 29, 33, 65–66, 68, 69,

72, 78–82, 93, 94, 96Linearization ........................................................18, 49, 65Linear regression ............................ 186, 187, 460, 466–470Line fitting ............................................................ 466–470Line-scan ........................................................334–336, 341Linker.. ........................................... 212–216, 302, 361, 367Lipid rafts ...................................................................... 411Lipofection ................................................................ 8, 337Lipophilic ligands .................................................. 139, 160Localisation ...................................... 31, 212, 217, 222, 280Luciferase ..................16, 299, 358, 360–366, 368, 374, 407Lysine scanning ............................................................... 87Lysis.... ........................................... 43, 45, 47, 50, 172, 245,

253–257, 299, 324, 325, 327, 348, 376, 378, 379, 401, 403, 431, 437

Lysosome ........................................................251, 252, 427

M

Mam2.. ........................................... 115, 122, 124, 126–128Mann–Whitney U test .................................................. 461Map3... .......................................................................... 115Mass spectrometry ......................... 216, 238, 239, 245–246,

319, 391, 394, 397, 400, 405–408Melting temperature ................................ 64, 80, 90, 92, 93,

96, 171, 182, 188Membrane ...................... 31, 41, 58, 82, 86, 89, 96, 99, 106,

119, 122–126, 129, 135–164, 195, 212, 214, 215, 217, 218, 222–227, 229, 231, 233–235, 242, 245, 248, 249, 251, 252, 255, 257, 258, 263–273, 288, 290, 297, 302, 303, 324, 325, 330, 331, 334–336, 339–341, 347–351, 353, 376–385, 389, 390, 395, 399, 403–404, 406, 407, 411–422, 425, 427, 428, 432, 439

Membrane preparation ..........................135–164, 263, 264, 376–381, 385, 407

Membrane raft markers ..........................414–415, 417–419Membrane rafts .............. 411, 412, 414–415, 417–419, 421Metabolic labeling ................................................. 348, 428MetaFluor ...................................... 307, 308, 310, 314, 340Metal ion bridge .............................................................. 88Methanethiosulphonate ................................................... 87Methylated DNA ............................................................ 96Methyl-b-cyclodextrin (mbCD) ................................... 422M-factor ........................................................................ 115mfold... ......................................................................171, 188mGluR ...........................................................204, 419, 420Microdomain .................................. 226, 411, 412, 415, 439Mifepristone .......................................................6, 7, 10, 15MIQE guidelines .................................................. 171, 184

ReceptoR Signal tRanSduction pRotocolS

477

Index

Models ..............................3, 4, 14–16, 23, 89, 99, 114, 147, 148, 150, 184, 187, 197, 198, 200, 202, 204–208, 228, 229, 234, 332, 400, 414, 428, 460, 466–470

mOrange ............................................................... 358–360Multiple cloning site ................................ 10, 28, 29, 62, 92Multiplex PCR .............................................................. 171Multiplicity of infection .................................................. 48Multivesicular body (MVB) .......................................... 252Muristerone A ................................................................. 15Muscarinic ................................... 26, 89, 96, 163, 195–197,

200, 208, 238–240, 249, 267, 305, 334, 336Mutagenesis..........................................40, 85–96, 101, 238Myc-tag ..............................................................26, 56, 256

N

N-ethylmaleimide (NEM) .................................... 253, 256Nitrocellulose............................31, 129, 242, 248, 255, 257,

347–349, 414, 418, 421, 437Non-linear regression ....................................148, 227–229,

232, 466, 468–470Non-orthogonal..................................................... 452, 454Non-parametric ...................... 446, 448, 449, 451, 460–462Non-raft proteins ...........................................414–415, 418Nonspecific binding........................... 14, 15, 137, 141–143,

149, 151–153, 155–163, 224, 232, 266, 269, 270, 350, 390, 393, 397, 432

Normal distribution ........................................445, 449–461Nucleofection ......................... 101, 102, 105, 108, 111, 337Null hypothesis ......................................449–452, 454, 455,

457–459, 463, 467, 468Numerical aperture (NA) ...............................226, 312, 434

O

Oil immersion ........................................314, 332, 333, 434Oligo dT.........................................................177–179, 190Oligomerization .................................................... 373–386Oligonucleotide ................................................. 89–92, 190One-site binding ........................................................... 144One-way analysis of variance ................................. 450–451Operational model ..........................................204, 206, 208Optical resolution .......................................................... 434Orthogonal ............................................................ 451–454Orthosteric ............................. 195–198, 200, 201, 204, 207Oscillation ............................................................. 281, 322

P

Parametric ...............444, 446, 448–451, 460–462, 465, 470Pearson correlation coefficient ....................................... 466Peptide affinity purification ................................... 389–408Peptide columns .............................................392–394, 396Pericams ................................................................ 278, 288Permeabilization ............................... 57, 289, 430, 433, 435Pertussis toxin (PTx) ............................................. 268, 271P-factor ..........................................................115, 124, 126

Pharmacophore .......212, 213, 215–217, 220, 221, 231, 232Pheromone ..................................... 115, 122, 124, 126, 129Phosphatidylinositol 4,5-bisphosphate (PIP2) .......330, 331,

338, 339, 342Phosphodiesterase ................................................. 298, 304Phospholipase C (PLC) ................................105, 108, 277,

330–334, 336, 340, 342Phospho-peptide map ................................................... 243Phosphor-imager ............................................242, 243, 245Phosphorylation .................... 100, 101, 104, 110, 237–249,

298, 367, 390Phospho-specific antibodies ...................238, 239, 246, 247Photoactivated localization microscopy (PALM) .......... 439Photobleaching ..........57, 293, 294, 305, 309, 313, 339, 341Phototoxicity ......................................................... 235, 313Plaque forming units (PFU) ............... 47, 48, 50, 89, 91, 93Plasmid .........................................4–7, 9, 11, 16, 18, 22, 23,

28–30, 33, 35, 40–46, 49, 50, 60–67, 69–72, 75, 76, 79–81, 89–96, 102–103, 108, 110, 111, 119–122, 128, 257, 319, 331, 350, 351, 377, 378, 380, 384, 403, 420

Plasmid linearization ............................................18, 49, 65Pluronic F–127 ...................................................... 278, 289Poly A tail .............................................................. 178, 179Polymerase ..............................59, 60, 67, 76, 80, 89, 91, 93,

95, 165–169, 176, 177, 350Polymerase chain reaction (PCR) ....................8, 10, 33, 59,

62, 64–69, 76–83, 88–90, 92–94, 96, 109, 165–192Polyvinylidene fluoride (PVDF) ............ 126, 129, 249, 324,

347–351, 437Ponasterone A ................................................................. 15[32P]orthophosphate .............................................. 239–242Post-hoc tests .........................................451, 454–456, 460Potency .............. 86, 204, 214, 221, 232, 342, 456, 458, 459pREP... ............................................................119, 123, 128Preparative gel ....................................................... 350, 353Primer.. .............................33, 64, 67–69, 73, 74, 76, 80–82,

89–95, 166–169, 171, 176–182, 188, 190, 191Primer-dimers ................................................167, 171, 177Probability ..................................58, 80, 120, 168, 177, 178,

309, 449, 450, 452, 455, 462–464, 468Probe-dependence ................................................. 202, 208Promoter .................................4–7, 9, 10, 19, 32–34, 40, 43,

49, 94, 122, 126, 128, 402, 428Proteasome .................................................................... 252Protein A affinity chromatography ................................ 404Protein A-sepharose ....................... 239, 240, 265, 268, 269Protein G ....................................................................... 274Protein kinase A (PKA) ........................................ 297–303Protein kinase C (PKC) ..................................330, 336, 341Protein–protein interactions ...................... 31, 56, 300, 347,

348, 358, 360Proteomics ..................................................................... 390Pull-down ............................... 318, 319, 323–324, 347–354Puromycin ..............................................................9, 11, 12

478 ReceptoR Signal tRanSduction pRotocolS

Index

Q

Quantification cycle....................................................... 166Quantitative PCR ..........................................183, 188, 191Quencher ................................................166, 168, 169, 182

R

Rabbit reticulocyte lysate ............................................... 354Radioligand ......................... 10, 14, 16, 135–164, 195–208,

217, 224, 232, 354, 403, 406, 428Radioligand binding ............................ 10, 14, 16, 135–164,

195–208, 217, 221, 232, 265, 403, 428, 466Random

hexamers .......................................................... 177, 179mutagenesis ....................................................86, 88, 89

Range.. ........................ 15, 54, 55, 57, 59, 75, 81–83, 85, 86, 128, 140, 160, 171, 179, 188, 195, 196, 199, 204, 206, 212, 213, 220–222, 224, 225, 228, 230, 231, 279, 281, 288, 302, 303, 313, 330, 333, 335, 338, 339, 354, 368, 374, 375, 389, 426–428, 438, 443, 446–448, 456, 458, 461, 466, 468

Rate constants ................................ 136, 137, 153, 154, 363Reading frame ....................................................29, 79, 126Real-time imaging ..........................................300–305, 331Receptor

number .....................................................135, 139, 428purification ...................................................... 245, 406reserve .......................................................106, 204, 232

Receptor-specific antibodies ...........................239, 406, 426Recombinase...................................................24, 28–30, 35Recombination ............................. 22, 42, 44–46, 49, 50, 88Recycling ........................................................251, 427, 428Reference gene .......................................176, 183–187, 191Region of interest (ROI) ....................... 223, 233, 283, 284,

294, 310, 311, 314, 333–336, 340, 341Regulator of G protein signaling (RGS) ........114, 323, 325Renaturation .................................................................. 348Renilla luciferase (Rluc) ................... 358, 359, 365, 369, 407Resonance energy transfer ......................212, 299, 367, 374Resource equation ................................................. 464, 465REST method ............................................................... 187Restriction

digest ......................................................................... 29endonuclease .......................................10, 60, 75–76, 78enzyme ..................43, 59, 64–65, 68, 72, 78, 90, 94, 96site. ............................................49, 62, 64, 78–80, 92, 94

Retrovirus ........................................................................ 40Reverse transactivator (rtTA) ............................................ 9Reverse transcription ......................................177, 179, 190Rho...... .................................................................. 317–327Rhod–2 .......................................................................... 293Rhotekin ......................................... 318, 319, 321, 323–326Ribonuclease .....................................................54, 349, 350Ribosomal RNA ............................................................ 175RIPA buffer ........................................ 27, 31, 239, 240, 248

Rluc2... ..............................................................358, 359, 368Rluc8... ............................. 358, 359, 363, 365, 366, 368, 369RNAi... ...................................................100, 101, 109, 110RNA integrity number ...................................173, 175, 176RNA interference .................................................. 101, 252RNA isolation ................................ 165, 172–174, 176, 177RNase... ..............................................59, 60, 70, 78, 82, 103,

110, 173–178, 182, 189–191Rosenthal plot ....................................................... 145, 159RT-qPCR .............................................................. 165–192Ryan-Einot-Gabriel-Welsch procedure ........................ 455

S

Saccharomyces cerevisiae ..............................................53, 124Saturation binding .......................... 136, 145, 199, 224, 353Scaffolding proteins ....................................................... 298Scanning spectrometry ................... 360, 362, 364, 368, 369Scatchard ....................................................................... 161Schild.. ................................................................... 198, 221Schizosaccharomyces pombe ........................................114–129SDM. See Site-directed mutagenesis (SDM)Selection .........................8–9, 11–12, 14, 19, 22, 23, 28–30,

33, 34, 36, 42, 55, 88, 92, 94, 111, 115, 119, 120, 122, 128, 183, 184, 222, 223, 234, 274, 349, 433, 444, 449

Sequencing .......... 29, 33, 70, 73, 92, 94, 171, 239, 244, 246Sheffé test ...................................................................... 455Short-hairpin (sh)RNA ................................................. 110SH-SY5Y ....................................... 107, 331, 332, 334, 336Shuttle plasmid .................................................... 42–44, 49Signaling platforms ....................................................... 412Signal peptide ..................................... 58, 66, 123, 129, 402Significance .................... 158, 412, 444, 449–460, 462–464siRNA. ................................................................... 101–110Site-directed mutagenesis (SDM) ..................... 85–96, 101SNAP... .............................................................24, 307, 426Snap-tag ................................................................ 377, 426Solubilization......................... 254, 257, 258, 265, 267–269,

271–273, 401, 404, 406, 407Southern blotting ...................................................... 23, 50Span.... ............................................ 147, 152, 329, 353, 467Spatial resolution ............................ 226, 283, 427, 434, 439Specific activity .......................................142, 158, 269, 354Specific binding ............................... 14, 137, 138, 142, 144,

146, 151–153, 159, 160, 197, 200, 202, 203, 224, 225, 232, 318

Sphericity .......................................................457, 459, 460Sphingomyelin .............................................................. 411Spin labelling ................................................................... 87Spinophilin ............................................................ 353, 354Stable expression ....................................................... 16, 21Stable isotope labelling with amino acids

in cell culture (SILAC) ...................................... 239Standard deviation (SD) ................................183, 365, 366,

445–447, 464, 465

ReceptoR Signal tRanSduction pRotocolS

479

Index

Standard error ................................................................ 446Sticky-ends ..............................60–62, 65, 66, 69, 78, 79, 82Stimulated emission decay (STED) microscopy ........... 439Stoke’s shift ................................................................... 220Streptavidin affinity chromatography ............................ 405Stripping......................................... 415, 421, 430, 431, 440Student Newman–Keuls test ......................................... 455Substituted cysteine accessibility method ........................ 87Subtype-selective ........................................................... 196Sucrose ............ 118, 119, 125, 376, 379, 413, 416, 417, 429Suicide enzymes ............................................................ 375Sulfinpyrazone ....................................................... 279, 290Summary statistics ................................................. 443–448Supercoiled DNA ............................................................ 10SybrGreen ............................................................. 166, 180

T

t1/2........ ..............................................................................138Tandem affinity purification (TAP) ...............395, 399–408Tandem tag .................................................................... 400TAP. See Tandem affinity purification (TAP)TAP tag .......................................... 400, 402, 403, 405, 406TaqMan®........................................................................ 166TATA box ................................................................ 10, 128Temperature cycling .......................................166, 167, 169Template .......................................64, 66, 69, 76, 79, 80, 86,

89, 91, 92, 94–96, 166–169, 171, 177, 178, 182, 184, 188, 349, 350

Terbium ................................................................. 374, 375Ternary complex ............................................................ 198Tet-Off .....................................................5, 7–9, 11, 15–17Tet-On .....................................................5, 7–9, 11, 15–17Tetracycline .................5, 7, 9, 15, 18, 22–25, 27, 29–31, 35Tetracycline repressor ................................................ 23, 29Tetracycline-responsive element (TRE) ............................ 5Tetratricopeptide repeat (TPR) ..................................... 318Thin layer chromatography (TLC) ............................... 243Threshold value ............................................................. 166Time-resolved fluorescent energy transfer

(TR-FRET) .............................................. 373–386TIRF. See Total internal reflection microscopy (TIRF)TLC. See Thin layer chromatography (TLC)TnT........................................................................ 348, 350Tobacco etch virus (TEV) protease ........400–402, 404, 407Total binding ................................... 87, 142, 143, 149, 151,

153, 155, 156, 158, 223Total internal reflection microscopy (TIRF) .........212, 278,

283, 287, 439TPR. See Tetratricopeptide repeat (TPR)Trafficking ................................... 54, 57, 58, 251, 252, 268,

338, 359, 361, 399, 425–440Transactivator ............................................................ 5, 7, 9Transfection ............................3, 6, 8, 10–12, 14, 16, 18, 19,

22–24, 29, 30, 32–35, 39, 41–47, 49, 50, 90, 95, 101–105, 107–111, 139, 187, 253, 254, 257, 302,

303, 307, 308, 331, 333, 337–339, 359, 361, 367, 377, 401–403, 406, 413, 416, 420

Transfection efficiency ........................... 18, 44, 49, 50, 104, 105, 108–110, 337

Transferrin .............................. 319, 323, 417–419, 421, 427Transformation .............................29, 65, 68, 69, 78, 80, 81,

83, 92, 96, 115–116, 119–122, 161, 293, 295, 445, 446, 448, 466

Transgene .................................................................. 14, 41Trans-Golgi network (TGN38) .................................... 427Transient expression .............................................. 334, 377Translocation .................. 106, 303, 330, 334–336, 339–342TRE. See Tetracycline-responsive element (TRE)T-Rex.. ................................................ 5, 7, 9, 15, 17, 21–36Triplet state ............................................213, 228, 234, 235Trypsin digestion ............................................394, 397, 405Tryptic digest ................................................................. 246Tryptic peptides ............................................................. 245t-test.... ..............................................................450, 455, 465Tubby... .......................................................................... 330Tukey test .............................................................. 455, 4682D electrophoresis ..........................................392, 394, 397Two-site binding ................................................... 147, 470Two-way analysis of variance ..................456, 458, 459, 470Type I error............................. 449, 450, 454, 455, 459, 463Type II error ...........................................449, 454, 463, 464

U

Ubiquitin ................................................252–254, 256–258

V

Variance ................................. 183, 366, 370, 446, 450–452, 457–459, 464, 468

Venus... ..................................... 57, 358, 359, 363–366, 369Viral titre ......................................................................... 47Virus.... ................................. 7, 39–41, 46–50, 56, 400, 402

W

Water immersion ............................................222, 227, 434Welch’s test .................................................................... 450Wilcoxon test ................................................................ 461Wilk–Shapiro test ......................................................... 448

Y

Yeast.... ............................ 7, 43, 53, 57–59, 73, 89, 113–130, 256, 257, 349, 390

Yellow fluorescent protein (YFP) ......................25, 57, 300, 301, 303, 305, 306, 309–314, 330, 341, 358, 360, 363, 374

Z

Zeocin........................................9, 12, 23, 24, 26–30, 33, 34Z’-factor ................................................................ 364–366Z-factor ......................................................................... 365