As key components of many cell signaling pathways, protein kinases are implicated in a broad variety of diseases, including cancers and neurodegenerative conditions, and offer considerable potential as tractable targets for therapeutic intervention. In Protein Kinase Protocols, a panel of highly skilled laboratory investigators describe both basic and more sophisticated methods for the analysis of kinase-mediated signaling cascades, with emphasis on the identification of proteins according to their interactive relationships and the analysis of their functional properties. Described in step-by-step detail, these readily reproducible techniques offer novices quick access to a complicated field, and provide more experienced investigators many novel time-saving ploys. Emphasis is given to the critical technical steps that are often omitted from methods published in the primary literature. There are also tips on potential pitfalls and copious notes on how to adjust the protocols to work in related systems.
Broad in its range of techniques and thoroughly detailed to help ensure experimental success, Protein Kinase Protocols offers both novice and experienced investigators powerful tools for understanding the functional roles of specific protein kinases within signaling cascades and for identification and evaluation of novel therapeutic targets.

Includes supplementary material: sn.pub/extras

Zusammenfassung
Rapid advances in both genome and proteome projects will enable scientists to catalogue the majority of proteins in key experimental species over the next 5-10 years. This text provides the methods and techniques utilized by experts.

Inhalt
Protein Kinase-Mediated Signaling Networks.- Cloning Protein Tyrosine Kinases by Screening cDNA Libraries with Antiphosphotyrosine Antibodies.- Immunoprecipitation and Western Blotting of Phosphotyrosine-Containing Proteins.- Two-Dimensional Phosphoamino Acid Analysis.- Two-Dimensional Phosphopeptide Mapping of Receptor Tyrosine Kinases.- Identification of the Sites of Phosphorylation in Proteins Using High Performance Liquid Chromatography and Mass Spectrometry.- Phosphorylation of Smad Signaling Proteins by Receptor Serine/Threonine Kinases.- Assays for Mitogen-Activated Protein Kinase (MARK) Subtypes and MARK Activating Protein Kinase-2 (MAPKAP K-2) Using a Common Cell Lysate.- JAK-Mediated Phosphorylation and Activation of STAT Signaling Proteins.- Assays for Glycogen Synthase Kinase-3(GSK-3).- Cyclin-Dependent Kinases and Cyclin-Dependent Kinase Inhibitors.- Protein Histidine Kinase.- Cloning and Expression of Recombinant HEXA-HIS Tagged ZAP-70 Using the Baculovirus System.- Analysis of Protein Kinase Subcellular Localization by Visualization of GFP Fusion Proteins.- Detection of Phosphorylation-Dependent Interactions by Far-Western Gel Overlay.- Purification of Tyrosine-Phosphorylated Proteins by Immunoaffinity Chromatography and Direct Cloning of Their cDNAs from Bacterial Expression Libraries.- Identification of Receptor Tyrosine Kinase (RTK) Substrates by the Cloning of ReceptorTargets (CORT) Strategy.- Identification of Receptor Tyrosine Kinase Associating Proteins Using the Yeast Two-Hybrid System.- cDNA Expression Cloning and Characterization of Phosphorylation Dependent Protein Interactors Using the Yeast Tribrid System.- Analysis of SH2 Domain - Phosphopeptide Interactions by Isothermal Titration Calorimetry and Surface Plasmon Resonance.- Cloning and Characterization of RTK Ligands Using Receptor-Alkaline Phosphatase Fusion Proteins.- Isolation and Characterization of Orphan-RTK Ligands Using an Integrated Biosensor Approach.