Cell biology assays : proteins

Preface vii List of Contributors ix I LABELING AND DETECTING PROTEINS 1. Protein Determination 3 2. Phosphopeptide Mapping: A Basic Protocol 13 3. Radioiodination of Proteins and Peptides 21 4. Protein Detection in Gels by Silver Staining: A Procedure Compatible with Mass Spectrometry 29 5. Fluorescence Detection of Proteins in Gels Using SYPRO Dyes 35 6. Autoradiography and Fluorography: Film-Based Techniques for Imaging Radioactivity in Flat Samples 47 II PROTEOMIC ANALYSES BASED ON PROTEIN SEPARATION 7. Free-Flow Electrophoresis 57 8. Gel-Based Proteomics: High-Resolution Two-Dimensional Gel Electrophoresis of Proteins. Isoelectric Focusing and Nonequilibrium pH Gradient Electrophoresis 65 9. High-Resolution Two-Dimensional Electrophoresis with Immobilized pH Gradients for Proteome Analysis 77 10. Two-Dimensional Difference Gel Electrophoresis: Application for the Analysis of Differential Protein Expression in Multiple Biological Samples 97 11. Image Analysis and Quantitation 109 12. Two-Dimensional Gel Profiling of Posttranslationally Modified Proteins by in vivo Isotope Labeling 121 III PROTEIN–PROTEIN AND PROTEIN–SMALL MOLECULE INTERACTIONS 13. Immunoprecipitation of Proteins under Nondenaturing Conditions 133 14. Nondenaturing Polyacrylamide Gel Electrophoresis as a Method for Studying Protein Interactions: Applications in the Analysis of Mitochondrial Oxidative Phosphorylation Complexes 141 15. Analysis of Protein–Protein Interactions by Chemical Cross-Linking 151 16. Peroxisomal Targeting as a Tool to Assess Protein–Protein Interactions 157 17. Biomolecular Interaction Analysis Mass Spectrometry 163 18. Ligand Blot Overlay Assay: Detection of Ca+2- and Small GTP-Binding Proteins 171 19. Modular Scale Yeast Two-Hybrid Screening 179 20. Affinity Electrophoresis for Studies of Biospecific Interactions: High-Resolution Two-Dimensional Affinity Electrophoresis for Separation of Hapten-Specific Polyclonal Antibodies into Monoclonal Antibodies in Murine Blood Plasma 191 21. Methods in Protein Ubiquitination 203 IV PROTEIN–DNA INTERACTIONS 22. Gel Mobility Shift Assay 219 23. DNA Affinity Chromatography of Transcription Factors: The Oligonucleotide Trapping Approach 231 V MASS SPECTROMETRY METHODS FOR DETERMINATION OF PROTEIN IDENTITY AND PROTEIN MODIFICATIONS 24. Protein Identification and Sequencing by Mass Spectrometry 243 25. Proteome Specific Sample Preparation Methods for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry 253 26. In-Gel Digestion of Protein Spots for Mass Spectrometry 263 27. Peptide Sequencing by Tandem Mass Spectrometry 269 28. Direct Database Searching Using Tandem Mass Spectra of Peptides 279 29. Identification of Proteins from Organisms with Unsequenced Genomes by Tandem Mass Spectrometry and Sequence- Similarity Database Searching Tools 289 30. Identification of Protein Phosphorylation Sites by Mass Spectrometry 301 31. Analysis of Carbohydrates/Glycoproteins by Mass Spectrometry 309 32. Stable Isotope Labeling by Amino Acids in Cell Culture for Quantitative Proteomics 325 33. Site-Specific, Stable Isotope Labeling of Cysteinyl Peptides in Complex Peptide Mixtures 337 34. Protein Hydrogen Exchange Measured by Electrospray Ionization Mass Spectrometry 345 35. Nongel-Based Proteomics: Selective Reversed-Phase Chromatographic Isolation of Methionine-Containing Peptides from Complex Peptide Mixtures 355 36. Mass Spectrometry in Noncovalent Protein Interactions and Protein Assemblies 361 Index 371