Small GTPases and Their Regulators, Part A: Ras Family

Preface
......Page 1
[1] Refolding and Purification of Ras Proteins......Page 7
[2] Purification of Baculovirus-Expressed Recombinant Ras and Rap Proteins......Page 17
[3] Purification of Recombinant Ras GTPase-Activating Proteins......Page 25
[4] Purification of Baculovirus-Produced Rap 1 GTPase-Activating Protein......Page 35
[5] Assays for Inhibitors of CAAX Farnesyltransferase in Vitro and in Intact Cells......Page 42
[6] Prenylation Analysis of Bacterially Expressed and Insect Cell-Expressed Ras and Ras-Related Proteins......Page 50
[7] Reticulocyte Lysate Assay for in Vitro Translation and Posttranslational Modification of Ras Proteins
......Page 64
[8] Carboxyl Methylation of Ras-Related Proteins......Page 69
[9] Use of Yeast for Identification of Farnesyltransferase Inhibitors and for Generation of Mutant Farnesyltransferases......Page 86
[10] Analysis of Intrinsic and CDC25-Stimulated Guanine Nucleotide Exchange of p21ras Nucleotide Complexes by Fluorescence Measurements
......Page 96
[11] Measurement of Nucleotide Exchange and Hydrolysis Activities in Immunoprecipitates
......Page 111
[12] Determination of Guanine Nucleotides Bound to Ras in Mammalian Cells......Page 119
[ 13] Purification of Baculovirus-Expressed Human Sos1 Protein
......Page 126
[14] Ras-Cdc25 and Rho-Dbl Binding Assays:  Complex Formation in Vitro......Page 130
[15] Analysis of Interaction between Ras and CDC25 Guanine Nucleotide Exchange Factor Using Yeast GAL4 Two-Hybrid System
......Page 136
[16] Measurement of Ras-Bound Guanine Nucleotide in Stimulated Hematopoietic Cells
......Page 150
[17] Measurements of GTP/GDP Exchange in Permeabilized Fibroblasts......Page 157
[18] Intrinsic and GTPase-Activating Protein-Stimulated Ras GTPase Assays
......Page 162
[19] Determination of Ras and GTPase-Activating Protein Interactions by Kinetic Competition Assay
......Page 172
[20] Phosphorylation-Dependent Complexes of p120 Ras-Specific GTPase-Activating Protein with p62 and p190......Page 180
[21] Analysis of Ras Protein Expression in Mammalian Cells
......Page 194
[22] Vaccinia Virus Expression of p21ras(Asn-17)
......Page 220
[23] Inducible Expression of Ras N17 Dominant Inhibitory Protein 
......Page 229
[24] Prenylation and Palmitoylation Analysis
......Page 236
[25] Immune Complex Kinase Assays for Mitogen-Activated Protein Kinase and MEK......Page 244
[26] Cell-Free Assay System for Ras-Dependent MEK Activation
......Page 256
[27] Preparation and Use of Semiintact Mammalian Cells for Analysis of Signal Transduction
......Page 264
[28] Mitogen-Activated Protein Kinase Activation by Scrape Loading of p21ras
......Page 272
[29] Assay and Expression of Mitogen-Activated Protein Kinase, MAP Kinase Kinase, and Raf
......Page 278
[30] Assay of MEK Kinases......Page 289
[31] Activation of Raf-1 by Ras in Intact Cells......Page 300
[32] Ras-Raf Complexes:  Analyses of Complexes Formed in Vivo
......Page 309
[33] Ras-Raf Complexes in Vitro......Page 322
[34] Ras-Raf Interaction:  Two-Hybrid Analysis
......Page 330
[35] Methods for Analyzing c-Jun Kinase......Page 341
[36] Use of Tyrosine-Phosphorylated Proteins to Screen Bacterial Expression Libraries for SH2 Domains
......Page 359
[37] Detection of SH3-Binding Proteins in Total Cell Lysates with Glutathione S-Transferase-SH3 Fusion Proteins:  SH3 Blot Assay
......Page 368
[38] Inhibition of Ras Function in Vitro and in Vivo Using Inhibitors of Farnesyl-Protein Transferase
......Page 377
[39] Rat Embryo Fibroblast Complementation Assay with ras Genes......Page 386
[40] Biological Assays for Ras Transformation......Page 392
[411 Ras-Mediated Transcription Activation: Analysis by Transient Cotransfection Assays......Page 409
[42] Oocyte Microinjection Assay for Evaluation of Ras-Induced Signaling Pathways......Page 423
[43] Mammalian Cell Microinjection Assay......Page 433
[44] Detection of Point Mutations in Ras in Tumor Cell Lines by Denaturant Gradient Gel Electrophoresis
......Page 439
[45] Diagnostic Detection of Mutant ras Genes in Minor Cell Populations
......Page 449
[46] RAS in Yeast: Complementation Assays for Test of Function
......Page 462
[47] Yeast Adenylyl Cyclase Assays......Page 465
[48] Purification of Human Neutrophil NADPH Oxidase Cytochrome b-558 and Association with Rap 1A
......Page 473
[49] Use of Yeast Two-Hybrid System to Evaluate Ras Interactions with Neurofibromin- GTPase-Activating Protein
......Page 485
[50] Screening Phage-Displayed Random Peptide Libraries for SH3 Ligands......Page 495
Author Index......Page 507
Subject Index......Page 532