Index

Actin, see Confocal immunofluorescence microscopy, cytoskeleton AFM, see Atomic force microscopy Anaphase-promoting complex, see

Ubiquitination Apoptosis, egg extract studies, advantages of system, 379, 380 caspase activation assay, 388, 392 cytochrome c release assay, 387,

388, 392 egg collection, 383 immunodepletion and protein addition studies, 391-393 interphase egg extracts, fractionation, cytosolic, light membrane, heavy membrane, and glycogen fractions, 389, 390, 392, 393 mitochondria isolation, 390 preparation, 385, 386, 392 materials, 380-383 nuclear morphology assay, 388 reconstitution assay, cytochrome c-induced apoptosis,

390, 391, 393 cytosolic and heavy membrane fractions, 390, 393 sperm chromatin demembranation for nuclear formation, 384, 385 spontaneous apoptosis induction, 379, 385, 386, 392 Atomic force microscopy (AFM), nuclear pore complex, calcium-mediated changes, 285 materials, 275

native complex imaging, 284, 285, 287 nuclei isolation from oocytes, 276,

285, 287 supports, 284, 287

temperature-dependent plugging and unplugging, 285

Borohydride, reduction of glutar-

aldehyde-fixed samples, 76

Calcium flux, oocytes, advantages of study, 103 confocal laser scanning microscopy, advantages, 110 framescan versus linescan imaging, 110, 112 instrumentation, 110 setup, 113, 118 dyes, 108, 109

fertilization release assay, 402, 403 flash photolysis of caged inositol 1,4,5-trisphosphate, 113, 114,119

materials for analysis, 104, 105, 115, 117,118

multiphoton laser scanning microscopy, calcium wave imaging after inositol 1,4,5-trisphos-phate induction, 94-96 oocyte preparation, defolliculation, 106 epithelial layer removal, 105 microinjection of caged inositol 1,4,5-trisphosphate, 106, 108,117, 118 surgery, 105

principles of epifluorescence microscopy and imaging, 109,110 total internal fluorescence microscopy imaging, advantages, 114 image acquisition, 115 instrumentation, 114, 115 materials, 104, 105 principles, 114

vitelline membrane stripping, 108

Capillary electrophoresis (CE), advantages in biological separations, 413

protein kinase A live cell assay, 426 spatial resolution, 414 subcellular sampling of oocytes, calibration curve preparation for inositol 1,4,5-trisphosphate, 421

capillary outlet positioning, 420 capillary preparation, 419 data analysis, 421 detector biosensor preparation and imaging, 420, 423 electrophoresis conditions, 419, 423

inositol 1,4,5-trisphosphate standards, 419, 420 materials, 414, 415, 422 oocyte preparation, 415, 423 sampling apparatus construction,

415, 417, 423 sampling volume determination, 418, 419

Caspase, activation assay, 388, 392 Cdc2, oocyte signaling studies, 438 cDNA, see Complementary DNA CE, see Capillary electrophoresis

Cell cycle, fertilization studies, 403 meiotic MI-MII transition studies in oocyte extracts, cell cycle progression monitoring, fluorescence microscopy, 451, 456

histone H1 kinase assay, 451, 452

membrane-permeabilized sperm nuclei preparation, 451, 456 cycling extract preparation from metaphase I oocytes, extract preparation, 450, 456 oocyte isolation, 448, 449, 456 synchronization at metaphase I, 450, 456 materials, 447

mitotic cycling extracts from activated eggs, 452-454, 456, 457 overview, 445-447 regulator proteolysis studies, metaphase-to-anaphase transition, immunodepletion and rescue assays, 229-231, 233 protein degradation assay, 228,

229, 232, 233 protein ubiquitination assay, 229, 233 mitosis exit and G1, 231, 232 Cell-free chromosomal DNA replication, demembranated sperm chromatin preparation, euthanization, 125 sperm concentration determination and storage, 127, 136

sperm extraction and purification,

125, 126, 136 testes extraction, 15 high-speed interphase extract preparation, 129 immunodepletion studies, 134-137 low-speed interphase extract preparation, 128, 129, 136 materials, 123-125 nuclear assembly extract assay limitations, 121-123 nucleoplasmic extract preparation, nuclear assembly and growth monitoring, 130, 133 nuclear assembly reactions, 129, 130

nuclei harvesting, 131, 136 nucleoplasm isolation, 131 oocyte maturation, ovulation, and collection, egg harvesting and dejellying, 128 egg laying induction with human chorionic gonadotropin, 127, 128

ovulation induction with pregnant mare serum gonadotropin, 127

origin unwinding studies, 133, 134 overview of egg extract system, 122, 123

replication assay in nucleoplasmic extract, 131, 132, 136 replication factor loading, 134 Chaperones, see Heat shock proteins ChIP, see Chromatin immunoprecipitation Chromatin assembly assays, materials, 142, 143, 145 micrococcal nuclease digestion assay, 144-146

oocyte preparation and single-stranded DNA microinjection, 143-145 overview, 139-142 supercoiling assay, 144, 145 Chromatin immunoprecipitation (ChIP), materials, 168-170 oocytes, microinjection, 172, 173, 178 preparation, 170, 171 thyroid hormone receptor transcriptional regulation analysis, 173, 174, 178 principles, 166, 167 tadpole development studies, chromatin preparation, 177, 179 immunoprecipitation, 177, 179 nuclei preparation, intestine and liver samples,

175, 177 tail samples, 175, 178 overview, 174, 175 tissue preparation, 175 triiodothyronine treatment, 175 thyroid hormone receptor coactivator and corepressor analysis overview, 167, 168 Chromosomal DNA replication, see Cell-free chromosomal DNA replication Chromosome condensation, egg extract studies, biochemical manipulation for functional studies, 470-472 cycled chromosome processing for immunofluorescence, 470 cytostatic factor extracts, features, 459, 460 preparation, crude extract, 462-464, 471

high-speed extract, 464, 465, 71

materials, 460-462 overview, 459, 460 time-lapse fluorescence microscopy, 469, 470, 472 CLSM, see Confocal laser scanning microscopy Complementary DNA (cDNA), full-insert sequences, 8, 10 library resources, 3 Confocal immunofluorescence microscopy, cytoskeleton, antibodies, secondary antibody selection, 77 storage, 77, 83, 84 types, 70, 71 bleaching, 76, 83 borohydride reduction of glutaral-dehyde-fixed samples, 76 clearing and mounting, 78-80 dyes, 72, 77 fixation, keratin filaments, 74 methanol fixation, 74 microtubules, 73, 74, 80, 83 hemisection for antibody penetration, 74-76 image acquisition, 82-84 instrumentation, 82 materials, 70, 83

oocyte/egg imaging advantages, 69 phalloidin staining of microtubules,

80, 81, 84 processing of samples, 77, 78, 83, 84 Confocal laser scanning microscopy (CLSM), calcium flux imaging in oocytes, advantages, 110 framescan versus linescan imaging, 110, 112

instrumentation, 110 setup, 113, 118 immunofluorescence, see Confocal immunofluorescence microscopy, cytoskeleton nucleocytoplasmic transport assay in oocytes, kinetic recordings, 263, 269 microchamber preparation and nucleus deposition, 263 nuclei isolation, 261-263, 267, 269

transport solution addition, 263 CSF extract, see Cytostatic factor extract

Cytochrome c, release assay for apoptosis detection, 387, 388, 392

Cytoskeleton imaging, see Confocal immunofluorescence microscopy, cytoskeleton Cytostatic factor (CSF) extract, see

Chromosome condensation; Fertilization; Spindle assembly; Ubiquitination

Defolliculation, calcium flux studies, 106 collagenase treatment, 33, 34 overview, 31, 32 sandpaper stripping, 34-36 voltage clamp studies, 336, 342 Xenopus tropicalis oocytes, 47, 48 DNA microarray, Affymetrix chip resources, 13 DNA replication, see Cell-free chromosomal DNA replication Drug assay, see Patch-clamp, oocytes; Voltage clamp, oocytes

Egg, apoptosis studies in extracts, see Apoptosis, egg extract studies chromosome condensation studies, see Chromosome condensation cytoskeleton imaging, see Confocal immunofluorescence microscopy, cytoskeleton DNA replication in extracts, see Cell-free chromosomal DNA replication fertilization studies in extracts, see

Fertilization Golgi apparatus disassembly reconstitution, see Golgi apparatus disassembly nuclear assembly assay, see Nuclear assembly assay nucleocytoplasmic transport assay, see Nucleocytoplasmic transport proprotein convertase studies, see

Proprotein convertases protein degradation studies, see

Ubiquitination spindle assembly studies, see Spindle assembly Electron microscopy (EM), nuclear pore complex, colloidal gold immunolabeling,

282-284, 287 cytoplasmic face of nuclear envelope, 276, 287 embedding and thin sectioning,

282, 287 materials, 274, 275, 285, 287 negative staining, 277, 278, 287 nuclei isolation from oocytes, 276, 285, 287

nucleoplasmic face of nuclear envelope, 276, 287 quick freezing/freeze-drying/ rotary metal shadowing, 278, 287 nucleocytoplasmic transport in oocytes, colloidal gold preparation, 306,

307, 311

gold conjugation of proteins, 307,

308, 312

gold conjugation of RNA, 308 materials, 305, 306 microinjection of colloidal gold conjugates, 309, 312 oocyte preparation, 308, 309, 312

overview and advantages, 301303, 305 sample preparation, 309-312 Electrophysiology, see Patch-clamp, oocyte; Voltage clamp, oocytes EM, see Electron microscopy Endoplasmic reticulum, multiphoton laser scanning microscopy imaging, 99 EST, see Expressed sequence tag Expressed sequence tag (EST), databases, 3

Fertilization, cell-free extract and raft studies, cytostatic factor extract preparation, 400 egg preparation, 399 fertilization, 399, 400, 409 mass spectrometry of raft-associated proteins, digestion and extraction of gel bands, 405, 406

expressed sequence tag database, 408, 409 identification of proteins, 405409

peptide mass fingerprinting, 407

product ion mass fingerprinting, 407 materials, 397, 398 raft preparation, 400, 409 signaling assays, calcium release assay, 402, 403 cell cycle transition, 403 mitogen-activated protein kinase phosphorylation, 402, 409, 410 phospholipase Cy immunodepletion, 403 phospholipase Cy phosphorylation, 402, 409, 410

protein tyrosine kinases in raft fractions, 402 Src overexpression effects on raft fractions, 403, 410 Src phosphorylation, 402, 409, 410

sperm preparation, demembranated sperm, 399 living sperm, 399 frog system advantages, 395, 396 Src activation, 396

Genetic map, resources, 10, 11 Genome physical map, resources, 11, 12

Genome sequencing, resources, 14-16 Xenopus tropicalis, 12, 13 Genomic libraries, resources, 11

Germinal vesicle breakdown (GVBD), assay, 49, 51 features, 446 Golgi apparatus disassembly, reconstitution from egg extracts and semi-intact Mardin-Darby canine kidney cells, biochemical analysis, immunodepletion, 362, 363 kinase inhibitors, 362 egg extract preparation, 359, 364 galactosyltransferase-green fluorescent protein reporter, cloning, 359 stable transfection, 359 incubation conditions, 360, 364 materials, 358, 359 morphometric analysis, 360 overview, 357, 358 semi-intact cell preparation, 360, 364 GVBD, see Germinal vesicle breakdown

Heat shock proteins (Hsps), classification, 213 protein refolding assays, luciferase activity assay after microinjection, heat-denatured protein, 218, 220

heat-denatured protein with

Hsp30C, 219, 220 luminometer settings, 217 nondenatured protein, 217, 218, 220 sample preparation, 217 materials, 214, 215 oocyte isolation and protein microinjection, 217, 220

overview, 214

recombinant Hsp30C preparation, bacteria expression and induction, 215, 220 purification, 215, 216, 220 Hsps, see Heat shock proteins

Immunoblot, see Western blot Immunodepletion, apoptosis studies in egg extract, 391-393

cell cycle regulator proteolysis and rescue assays in egg extracts, 229-231, 233 cell-free chromosomal DNA

replication studies, 134-137 Golgi apparatus disassembly studies,

362, 363 nuclear import studies in reconstituted nuclei, 298, 299 oocyte signaling studies, 441 phospholipase Cy studies in fertilization, 403 Immunofluorescence microscopy, see Confocal immunofluorescence microscopy, cytoskeleton Inositol 1,4,5-trisphosphate (IP3), caged compound studies, see

Calcium flux, oocytes subcellular sampling and analysis of oocytes, see Capillary electrophoresis Intermediate filament, see Confocal immunofluorescence microscopy, cytoskeleton IP3, see Inositol 1,4,5-trisphosphate

Karyopherins, nucleocytoplasmic transport mediation, 243-246

Keratin filament, see Confocal immunofluorescence microscopy, cytoskeleton

Lipid rafts, see Fertilization

MAPK, see Mitogen-activated protein kinase

Mardin-Darby canine kidney cell, see

Golgi apparatus disassembly Mass spectrometry (MS), raft-associated proteins, digestion and extraction of gel bands, 405, 406 expressed sequence tag database, 408, 409

identification of proteins, 405-409 peptide mass fingerprinting, 407 product ion mass fingerprinting, 407 Mechanosensitive channels, see Patch-

clamp, oocytes Membrane transplantation, oocytes, cytoplasmic injection of membranes, 354

materials, 348, 349, 353 membrane preparation for transplantation, cell lines, 350, 352, 343 mammalian brain tissue, 349, 350, 353 oocyte preparation, 349 rationale, 347, 348, 353 voltage clamp recordings, 353, 354 Messenger RNA polyadenylation, cytoplasmic polyadenylation element, 184 oocyte assays, materials, 185, 186 RNA polyadenylation changes, DNA template generation, 194

gel electrophoresis and autoradiography, 195 overview, 192-194, 196 radiolabeled RNA probe generation, 194, 196 RNA extraction, 194, 195 3'-untranslated region translational effects, DNA template generation for in vitro transcription, 189,

dual-luciferase reporter assay, 192

overview, 186, 188, 189, 195 RNA microinjection, 190-192,

runoff assay and messenger RNA quantification, 189, 190, 195, 196 overview, 183, 184 translational control, 184, 185 Messenger RNA splicing, see Premessenger RNA splicing Micrococcal nuclease, see Chromatin assembly assays Microinjection, caged inositol 1,4,5-trisphosphate into oocytes, 106, 108, 117, 118

colloidal gold conjugates into oocytes, 309, 312 DNA into oocytes for chromatin immunoprecipitation assay, 172, 173, 178 membrane transplantation, 352-354 pre-messenger RNA splicing studies, antisense U2 DNA, 156, 161, 162 needle preparation, 156 radiolabeled pre-messenger RNA, 157, 158, 162

rescuing U2 small nuclear RNA, 157, 162 proteins into oocytes, 217, 220 receptor and channel constructs into oocytes, 337, 338, 342, 343 RNA into oocytes, 190-192, 196 single-stranded DNA into oocytes, 143-145

viral RNA into oocytes, 370, 371, 377

Microtubule, see Confocal immunofluorescence microscopy, cytoskeleton Mitochondria, isolation from eggs, 390 multiphoton laser scanning microscopy imaging, 96, 98

Mitogen-activated protein kinase (MAPK), oocyte signaling studies, 437, 438 phosphorylation assay in fertilization, 402, 409, 410 Molecular chaperones, see Heat shock proteins MPLSM, see Multiphoton laser scanning microscopy MS, see Mass spectrometry Multiphoton laser scanning microscopy (MPLSM), advantages in oocyte imaging, 87, 88 calcium wave imaging after inositol 1,4,5-trisphosphate induction, 94-96 endoplasmic reticulum imaging, 99 image acquisition, 93, 94 instrumentation, 88, 90, 91 limitations in oocyte imaging, 99 materials, 92

mitochondria imaging, 96, 98

pigmented versus albino oocyte studies, 92, 93 principles, 90-92 Mutagenesis, resources, 13, 14

Neurotransmitter receptors, see Voltage clamp, oocytes Nuclear assembly assay, egg extract assays, demembranated sperm chromatin preparation, 294, 295 fractionation of extracts, 292,

293,295 frog priming and egg-laying induction, 291, 292, 299 materials, 290, 291 nuclei reconstitution, 295, 296, 299 overview, 289, 290 limitations, 121-123 Nuclear envelope, see

Nucleocytoplasmic transport Nuclear pore complex, see

Nucleocytoplasmic transport Nucleocytoplasmic transport, diffusion control, 236, 237 egg extract assays, demembranated sperm chromatin preparation, 294, 295 fractionation of extracts, 292,

293,295 frog priming and egg-laying induction, 291, 292, 299 import assays, immunodepletion studies, 298, 299

reconstituted nuclei, 297, 298 substrate preparation, 297 materials, 290, 291 nuclei reconstitution, 295, 296, 299

overview, 289, 290 history of study, 235, 236, 260 karyopherin-mediated transport, 243-246

nuclear envelope structure, 236, 237 nuclear localization signals, 243-246 nuclear pore complex, atomic force microscopy, calcium-mediated structural changes, 285 materials, 275

native complex imaging, 284,

285, 287 nuclei isolation from oocytes,

276, 285, 287 supports, 284, 287 temperature-dependent plugging and unplugging, 285

electron microscopy, colloidal gold immunolabeling,

282-284, 287 cytoplasmic face of nuclear envelope, 276, 287 embedding and thin sectioning,

282, 287 materials, 274, 275, 285, 287 negative staining, 277, 278, 287

nuclei isolation from oocytes,

276, 285, 287 nucleoplasmic face of nuclear envelope, 276, 287 quick freezing/freeze-drying/ rotary metal shadowing, 278, 287 inert molecule permeability, 242,

243,252 nucleoporin types and structures, 240-242

structure, 238, 240, 242, 273, 274 translocation mechanism models, affinity gradient model, 250 oily spaghetti model, 250 reduction-of-dimensionality model, 252 selective-phase model, 250, 252

Stewart model, 250 virtual gating model, 250 NXF-mediated export of ribonucleoproteins, 246-248 oocyte assays, confocal laser scanning microscopy, kinetic recordings, 263, 269 microchamber preparation and nucleus deposition, 263 nuclei isolation, 261-263, 267, 269

transport solution addition, 263 electron microscopy, colloidal gold preparation, 306,

307,311 gold conjugation of proteins,

307,308,312 gold conjugation of RNA, 308 materials, 305, 306 microinjection of colloidal gold conjugates, 309, 312 oocyte preparation, 308, 309, 312

overview and advantages, 301303, 305 sample preparation, 309-312 materials, 260, 261 optical single transport recording, chamber preparation, 265 data evaluation, 267, 270, 271 kinetic recordings, 266, 267, 269

nuclear envelope preparation,

265, 270 nuclei attachment to test compartments, 265, 270 nuclei isolation, 265 principles, 260, 269, 270 overview, 260 NXF proteins, ribonucleoprotein export mediation, 246-248

Oocyte, anatomy of developed oocyte, 19, 20 calcium flux imaging, see Calcium flux, oocytes cell cycle studies, see Cell cycle chromatin assembly, see Chromatin assembly assays chromatin immunoprecipitation, see Chromatin immunoprecipitation electrophysiology, see Patch-clamp, oocytes; Voltage clamp, oocytes isolation and enucleation, defolliculation, collagenase treatment, 33, 34 overview, 31, 32 sandpaper stripping, 34-36 enucleation, functional studies, 37, 39, 40 Western blot analysis, 37, 39, 40 materials, 32, 33 ovarian tissue isolation, 33, 39 maturation and ovulation, 26, 27,

435, 436 membrane transplantation, see

Membrane transplantation, oocytes messenger RNA polyadenylation assay, see Messenger RNA polyadenylation microinjection, see Microinjection multiphoton laser scanning microscopy, see Multiphoton laser scanning microscopy nucleocytoplasmic transport studies, see Nucleocytoplasmic transport oogenesis, overview, 20, 21 stage I, 22 stage II, 22 stage III, 22, 23 stage IV, 23 stage V, 23 stage VI, 24 ovary, see Ovary protein expression, see also T7 RNA polymerase expression system, advantages, 55 applications, 55, 56 approaches, 56 receptors and channels, see Voltage clamp, oocytes protein kinase A assay, see Protein kinase A protein refolding assay, see Heat shock proteins RNA splicing, see Pre-messenger

RNA splicing RNA virus replication, see RNA

virus replication, oocytes signaling studies, advantages, 436, 437 difficulties, 437 examples, 437, 438 extract preparation, 439-442 immunodepletion, 441 materials, 438 oocyte harvesting, 439, 441 pharmacological agents, 441

stimuli, 440, 441 subcellular sampling, see Capillary electrophoresis vitellogenesis, 24, 25 Xenopus tropicalis oocytes, see Xenopus tropicalis Optical single transport recording

(OSTR), nucleocytoplasmic transport assay in oocytes, chamber preparation, 265 data evaluation, 267, 270, 271 kinetic recordings, 266, 267, 269 nuclear envelope preparation, 265, 270 nuclei attachment to test compartments, 265, 270 nuclei isolation, 265 principles, 260, 269, 270 OSTR, see Optical single transport recording

Ovary, anatomy, 17-19

oocyte maturation and ovulation, 26, 27

oogenesis, see Oocyte steroidogenesis, 25, 26

Patch-clamp, oocytes, drug assays, automation, 332 defolliculation, 336, 342 expression of channels and receptors, incubation, 338 microinjection, 337, 338, 342, 343

materials, 332-335 recording, 339, 341, 344 voltage-gated channel mechanosensitivity, channel expression density, 316, 317

dose-response studies, 323, 326 endogenous currents, gadolinium carbonate artifacts, 325

overview, 323, 324 stretch-activated cation nonselective current, 323, 325, 326 materials, 316 mechanostimulator, 321 micromanipulator adjustments and oocyte flattening, 319 microscopy, 317, 319 oocyte advantages versus mammalian cells, 316 overview, 315, 316 patch history, 319, 320 recording pipets, 320 series resistance effect, 326 single-channel measurements, 323

solutions, 320, 321 stretch/clamp techniques, 321, 322

Peptide mass fingerprinting, see Mass spectrometry Pesticide assay, see Patch-clamp, oocytes; Voltage clamp, oocytes Phospholipase Cy immunodepletion studies in fertilization, 403 phosphorylation assay in fertilization, 402, 409, 410 PKA, see Protein kinase A Poliovirus, see RNA virus replication, oocytes Pre-messenger RNA splicing, machinery, 149 model systems, 150

oocyte studies, advantages, 150, 161 materials, 150-153 microinjection, antisense U2 DNA, 156, 161, 162

needle preparation, 156 radiolabeled pre-messenger

RNA, 157, 158, 162 rescuing U2 small nuclear RNA, 157, 162 nuclear isolation, 158, 159, 162 oocyte preparation, anesthesia, 155, 161 separation and isolation, 155,

156,161 surgery, 155, 161 overview, 150 RNA synthesis, pre-messenger RNA, 154 U2 small nuclear RNA, 153, 154,161 splicing assay, 159, 160 Proprotein convertases, classification, 199 egg extract translation system studies, autocatalytic processing, 204, 210 complementary RNA preparation,

201, 209 endogenous activity, 204-206 glycosylation inhibition, 202-204 materials, 200, 201 overview, 200

pH-dependent aggregation, 206, 207

translation, 202, 209, 210 transmembrane domain identification in protease protection, 207-209

structure, 199, 200 7B2 as substrate, 205, 206 Proteases, see Proprotein convertases Protein degradation, see Ubiquitination Protein kinase A (PKA), live cell assays, capillary electrophoresis assay, 426

fluorescence resonance energy transfer-based substrates, 425-426 Myr-HA-ß2AR-C as substrate, COS7 cell assay, 428, 429, 432 materials, 426, 427 oocyte assay, 427, 428, 432 overview, 426 phosphorylation analysis with Western blot, 429-432 subunits, 425 Protein refolding assay, see Heat shock proteins

Rafts, see Fertilization

RanGTP, nucleocytoplasmic transport role, 243-246, 252 RNA splicing, see Pre-messenger RNA splicing

RNA virus replication, oocytes, infectious viral particle production,

374, 375, 378 internal ribosome entry site-dependent translation requirements, 375, 376, 378 materials, 368, 369 overview, 367, 368 RNA replication assays, all newly-synthesized RNA, 372, 377

negative-strand RNA synthesis, 372-374, 377

translation of genomic RNA, microinjection of viral RNA, 370,

371, 377 poliovirus RNA purification from virions, 369, 370

Semi-intact cell, see Golgi apparatus disassembly Small nuclear RNA, see Pre-messenger

RNA splicing Spindle assembly, egg extract studies, biochemical manipulation for functional studies, 470472

cycled spindle reactions and anaphase, 466-468 cytostatic factor extracts, features, 459, 460 preparation, crude extract, 462-464, 471 high-speed extract, 464, 465, 71

half-spindle assembly reactions, 465,

466, 471, 472 materials, 460-462 overview, 459, 460 spin-down processing for immunofluorescence, 468, 472

Xenopus tropicalis, 49, 50 Src, fertilization activation, 396 phosphorylation assay in fertilization, 402, 409, 410 overexpression effects on raft fractions in fertilization, 403, 410 Stretch, see Patch-clamp, oocytes Subcellular sampling, see Capillary electrophoresis

T7 RNA polymerase expression system, advantages, 56

cytoskeleton imaging, see Confocal immunofluorescence microscopy, cytoskeleton expression plasmids, cDNA constructs, 58 vector, 58 ion channel expression, 57, 65 materials, 57, 58 metabolic labeling, 64, 65 oocyte, injection with RNA polymerase/

cDNA mixture, 64 maintenance, 61, 64 preparation, 61 RNA polymerase preparation, activity assay, 61 bacterial transformation and induction, 58, 59 cell disruption, 59, 60 dialysis, 60 gel electrophoresis, 60 purification, 60 storage, 61 yield, 61 validation, 65, 66 Tadpole, see Chromatin immunoprecipitation Thyroid hormone receptor, see Chromatin immunoprecipitation TIRFM, see Total internal fluorescence microscopy Total internal fluorescence microscopy (TIRFM), calcium flux imaging in oocytes, advantages, 114 image acquisition, 115 instrumentation, 114, 115

materials, 104, 105 principles, 114

vitelline membrane stripping, 108 Translation assay, see Messenger RNA

polyadenylation Two-dimensional gel electrophoresis, protein kinase A assay in live cells, COS7 cell assay, 428, 429, 432 materials, 426, 427 oocyte assay, 427, 428, 432 overview, 426 Two-electrode voltage clamp, see Voltage clamp, oocytes

Ubiquitination, anaphase-promoting complex, cell cycle regulators proteolyzed in mitosis exit and G1, 231, 232

ubiquitin ligase activity, 224 egg extract studies, cell cycle regulators proteolyzed in metaphase-to-anaphase transition, immunodepletion and rescue assays, 229-231, 233 protein degradation assay, 228,

229, 232, 233 protein ubiquitination assay, 229, 233 cytostatic factor extract preparation, 226, 228 interphase extract preparation,

226-228, 232 materials, 224-226 enzymes, 224 UniGene clusters, resources, 8, 14

Vitellogenesis, process, 24, 25

Voltage clamp, oocytes, drug assays, automation, 332 defolliculation, 336, 342 expression, channels and receptors, incubation, 338 microinjection, 337, 338, 342, 343

materials, 332-335 neurotransmitter receptor ligands, agonists, 341, 344 antagonists, 341, 342 two-electrode voltage clamp, electrode preparation, 339, 343 recording, 339, 343, 344 rig wiring, 338 overview, 331, 332 transplanted membrane recordings, 353, 354

Western blot, cytochrome c release assays, 387,

388, 392 mitogen-activated protein kinase phosphorylation in fertilization, 402, 409, 410 oocyte enucleation for analysis, 37, 39, 40

phospholipase Cy phosphorylation in fertilization, 402, 409, 410

protein kinase A assays in live cells, Myr-HA-P2AR-C as substrate, COS7 cell assay, 428, 429, 432

materials, 426, 427 oocyte assay, 427, 428, 432 overview, 426 phosphorylation analysis, 429432

Src phosphorylation in fertilization,

402, 409, 410 Xenopus tropicalis oocyte lysate preparation, 50

Xenopus tropicalis, advantages in developmental studies,

43, 44 genetic resources, 1-16 genome sequence, 12, 13 oocyte studies, defolliculation, 47, 48 germinal vesicle breakdown assay, 49, 51 isolation, 46, 47 lysate preparation, histone H1 kinase assay, 51 Western blot, 50 materials, 44, 45, 51 maturation induction, 45, 46, 48 spindle formation assays, 49, 50 popularity of use, 2, 3, 13

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