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Protein Phosphatase Protocols presents a broad range of protocols for the study of protein phosphatases, all written by experts and innovators from phosphatase laboratories around the world. This volume is a compendium of resources for the study of protein phosphatases and their potential as drug targets. Experimental methodologies are taken from proteomics, bioinformatics, genomics, biochemistry, RNAi, and genetics. Included protocols utilize several model organisms, including yeast, Drosophila, and various plant and human cells. Protein Phosphatase Protocols provides a concise introduction to protein phosphatase research, with particular attention to phosphatase families. Assays for identification, quantification, purification, and functional characterization are presented throughout in specific detail.
Table des matières
Analysis of Protein Phosphatases.- A Brief Introduction to the Protein Phosphatase Families.- Small-Molecule Inhibitors of Ser/Thr Protein Phosphatases.- Synthesis and Use of the Protein Phosphatase Affinity Matrices Microcystin-Sepharose and Microcystin-Biotin-Sepharose.- Utilizing Protein Phosphatase Inhibitors to Define PP2A as a Regulator of Ataxia-Telangiectasia Mutated.- An Automated Fluorescence-Based Method for Continuous Assay of PP2A Activity.- An In Vivo Assay to Quantify Stable Protein Phosphatase 2A (PP2A) Heterotrimeric Species.- Mutagenesis and Expression of the Scaffolding A? and A? Subunits of PP2A.- Isolation and Characterization of PP2A Holoenzymes Containing FLAG-Tagged B Subunits.- Purification of PP2A Holoenzymes by Sequential Immunoprecipitation with Anti-Peptide Antibodies.- Purification of PP2Ac from Bovine Heart.- Visualization of Intracellular PP1 Targeting Through Transiently and Stably Expressed Fluorescent Protein Fusions.- Yeast Two-Hybrid Screens to Identify Drosophila PP1-Binding Proteins.- Identification of Cellular Protein Phosphatase-1 Regulators.- Assay for Three-Way Interaction of Protein Phosphatase-1 (Glc7) With Regulatory Subunits Plus Phosphatase Inhibitor-2.- Phosphorylation of the Protein Phosphatase Type 1 Inhibitor Protein CPI-17 by Protein Kinase C.- Purification of Smooth Muscle Myosin Phosphatase Using a Thiophosphorylated Myosin Light-Chain-Affinity Resin.- Proteins Interacting With Saccharomyces cerevisiae Type 1 Protein Phosphatase Catalytic Subunit Identified by Single-Step Affinity Purification and Mass Spectrometry.- Expression of Protein Histidine Phosphatase in Escherichia coli, Purification, and Determination of Enzyme Activity.- The Use of RNA Interference to Analyze Protein Phosphatase Function in MammalianCells.- Recognition of a PP2C Interaction Motif in Several Plant Protein Kinases.- Use of Yeast Genetic Tools to Define Biological Roles of Novel Protein Phosphatases.- Targeting of PP2C in Budding Yeast.- Phosphatase Targets in TOR Signaling.- Functional Characterization of Small CTD Phosphatases.- Genome-Scale Discovery and Characterization of Class-Specific Protein Sequences.- Yeast Substrate-Trapping System for Isolating Substrates of Protein Tyrosine Phosphatases.
Résumé
Protein Phosphatase Protocols presents a broad range of protocols for the study of protein phosphatases, all written by experts and innovators from phosphatase laboratories around the world. This volume is a compendium of resources for the study of protein phosphatases and their potential as drug targets. Experimental methodologies are taken from proteomics, bioinformatics, genomics, biochemistry, RNAi, and genetics. Included protocols utilize several model organisms, including yeast, Drosophila, and various plant and human cells. Protein Phosphatase Protocols provides a concise introduction to protein phosphatase research, with particular attention to phosphatase families. Assays for identification, quantification, purification, and functional characterization are presented throughout in specific detail.
