Regulatory protein modification : techniques and protocols

Analysis of Protein Phosphorylation in Intact Cells and Extracts, S. Ivar Walaas and Anne Carine Ostvold. Introduction. Investigating Protein Phosphorylation Systems. General Considerations. Experimental Pitfalls. Phosphorylation of Proteins in Intact Preparations. General Considerations. Intact Brain. Brain Slices. Isolated Cells and Nerve Terminals. Phosphorylation of Proteins in Cell-Free Preparations. General Considerations. Labeling and Stimulation of Cell-Free Preparations. Analysis of Phosphoproteins. General Considerations. Analysis of Protein Kinases. General Considerations. Analysis of Protein Kinase Activity In Vitro. Analysis of Specific Protein Kinases In Vitro. Analysis of Protein Kinase Activity in Intact Cells. Analysis of Protein Kinase Activity by Renaturation In Vitro. References. Analysis of Protein Dephosphorylation in Intact Cells and Extracts, Shelley Halpain. Introduction. Serine/Threonine Protein Phosphatases. Regulation of Phosphatase Activity. Measurement of Phosphatase Activity in Neural Tissue. Protocols. Measurement of Protein Phosphatase Activity in Extracts. Assaying the Phosphorylation State of Phosphoprotein Substrates in Tissue. Pharmacological Tools for Identifying Phosphatases that Target Proteins in Intact Cells. Dephosphorylation vs Proteolysis: A Caveat. Acknowledgments. References. Protein Phosphorylation and Dephosphorylation in Isolated Nerve Terminals (Synaptosomes), Talvinder S. Sirha. Introduction. Isolated Nerve Terminals (Synaptosomes). Background (Historical). Preparation of Synaptosomes and Purification by Percoll Gradients. Protein Phosphorylation/Dephosphorylation in Synaptosomes. Background. Prelabeling Synaptosomes with [32P] Orthophosphate. Modulators of Protein Kinases and Phosphatases. Analysis of Synaptosomal Phosphoproteins. Materials. Final Remarks. Acknowledgments. References. Protein Kinase and Phosphatase Inhibitors: Applications in Neuroscience, Hugh C. Hemmings, Jr. Introduction. Protein KinaseInhibitors. Cyclic Nucleotide-Dependent Protein Kinase Inhibitors. Ca2+/Calmodulin-Dependent Protein Kinase Inhibitors. Nonselective Protein Kinase Inhibitors (Staurosporine and Analogs). PKC Inhibitors. Cyclin-Dependent Kinase Inhibitors. Mitogen-Activated Protein Kinase (MAPK) and MAPK Kinase (MEK) Inhibitors. Protein-Tyrosine Kinase Inhibitors. Protein Phosphatase Inhibitors. Protein-Serine/Threonine Phosphatase Inhibitors . Protein-Tyrosine Phosphatase Inhibitors. Acknowledgments. Appendix I: Sources of Protein Kinase Inhibitors. Appendix II: Sources of Protein Phosphatase Inhibitors. References. Phosphorylation State-Specific Antibodies, Andrew J. Czernik, Jeffrey Mathers, and Sheenah M. Mische. Introduction. Phosphorylation State-Specific Antibody Production. Design of Phosphopeptides. Preparation of Phosphopeptides. Phosphopeptide Conjugation and Immunization. Primary Screening of Antiserum for Phosphorylation State-Specificity. Affinity Purification of Antisera. Advantages, Limitations, and Applications of Phosphorylation State-Specific Antibodies. Acknowledgments. References. Protein Tyrosine Phosphorylation, Pascal Derkinderen and Jean-Antoine Girault. Introduction. Overview of Tyrosine Phosphorylation in the Nervous System. Protein-Tyrosine Kinases. Protein-Tyrosine Phosphatases (PTPs). Biochemical Consequences of Tyrosine Phosphorylation. Neuronal Proteins Phosphorylated on Tyrosine. Regulation of Protein Tyrosine Phosphorylation in the Nervous Tissue. Methods to Study Protein Tyrosine Phosphorylation and Dephosphorylation in Nervous Tissue. Overview of the Methods Available. Model Systems for Studying the Regulation of Tyrosine Phosphorylation. Antiphosphotyrosine Antibodies. Immunoblotting with Antiphosphotyrosine Antibodies. Immunoprecipitation. In Vitro Protein Kinase Assays Using Immunoprecipitates. References. Identification of Posttranslational Modification Sites by Site-Directed Mutagenesis, James A. Bibb and Edgar F. da Cruz e Silva. Introducti