Protein Tyrosine Phosphatases

This book provides coverage, methodology, and laboratory protocols on the more essential aspects of protein tyrosine phosphatase (PTP) function and regulation, including the use of standardized in vitro functional assays, suitable cell systems, and animal and microorganism models. Chapters covering state-of-the-art technical approaches suitable to decipher the physiologic roles of PTPs, and their involvement in tissue-specific functions, are also included, which will be of utility for both newcomers and experienced researchers in the field of tyrosine- and phosphoinositide- phosphorylation/dephosphorylation. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Protein Tyrosine Phosphatases: Methodsand Protocols aims to aid researchers in better defining the common and individual features of the PTP family members and translating this knowledge into PTP-based therapy for human disease.

Includes cutting-edge methods and protocols involving the essentials of PTP function and regulation Provides step-by-step detail essential for reproducible results Contains key notes and implementation advice from the experts Includes supplementary material: sn.pub/extras

Inhalt

The Extended Family of Protein Tyrosine Phosphatases.- Global RT-PCR and RT-qPCR Analysis of the mRNA Expression of the Human PTPome.- Expression, Purification, and Kinetic Analysis of PTP Domains.- Peptide Microarrays for Real Time Kinetic Profiling of Tyrosine Phosphatase Activity of Recombinant Phosphatases and Phosphatases in Lysates of Cells or Tissue Samples.- Tailor-Made Protein Tyrosine Phosphatases: In Vitro Site-Directed Mutagenesis of PTEN and PTPRZ-B.- Assays to Measure PTEN Lipid Phosphatase Activity In Vitro from Purified Enzyme or Immunoprecipitates.- Assessing the Biological Activity of the Glucan Phosphatase Laforin.- Discovery and Evaluation of PRL Trimer Disruptors for Novel Anticancer Agents.- Analyzing Pseudophosphatase Function.- Crystallization of PTP Domains.- NMR Spectroscopy to Study MAP Kinase Binding to MAP Kinase Phosphatases.- Visualizing and Quantitating the Spatiotemporal Regulation of Ras/ERK Signaling by Dual-Specificity Mitogen-Activated Protein Phosphatases (MKPs).- In Situ Proximity Ligation Assay (In Situ PLA) to Assess PTP-Protein Interactions.- Use of Dominant Negative/Substrate Trapping PTP Mutations to Search for PTP Interactors/Substrates.- Detection and Identification of Ligands for Mammalian RPTP Extracellular Domains.- Production of Osteoclasts for Studying Protein Tyrosine Phosphatase Signaling.- Functional Analysis of Protein Tyrosine Phosphatases in Thrombosis and Haemostasis.- Functional Analysis of Dual-Specificity Protein Phosphatases in Angiogenesis.- Studying Protein-Tyrosine Phosphatases in Zebrafish.- Live staining of Drosophila Embryos with RPTP Fusion Proteins to Detect and Characterize Expression of Cell-Surface RPTP Ligands.- Methods to Study Protein Tyrosine Phosphatases Acting on Yeast MAPKs.