Protein Kinase ProtocolsAlastair D. Reith Springer Science & Business Media, 02.02.2008 - 365 Seiten 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. |
Inhalt
1 | |
Cloning Protein Tyrosine Kinases by Screening cDNA Libraries | 21 |
Immunoprecipitation and Western Blotting of Phosphotyrosine | 39 |
Peter BlumeJensen and Tony Hunter | 49 |
TwoDimensional Phosphopeptide Mapping of Receptor | 67 |
Identification of the Sites of Phosphorylation in Proteins Using High | 87 |
Phosphorylation of Smad Signaling Proteins by Receptor Serine | 107 |
a Common Cell Lysate | 121 |
Analysis of Protein Kinase Subcellular Localization | 195 |
by FarWestern Gel Overlay | 209 |
Purification of TyrosinePhosphorylated Proteins | 221 |
by the Cloning of Receptor Targets CORT Strategy | 239 |
Identification of Receptor Tyrosine Kinase Associating Proteins | 251 |
cDNA Expression Cloning and Charaterization of Phosphorylation | 271 |
Analysis of SH2 DomainPhosphopeptide Interactions | 295 |
Cloning and Characterization of RTK Ligands Using Receptor | 313 |
JAKMediated Phosphorylation and Activation of STAT Signaling | 131 |
Darren Cross | 147 |
Harry R Matthews and Karina Chan | 171 |
Cloning and Expression of Recombinant HEXAHIS Tagged | 183 |
Isolation and Characterization of OrphanRTK Ligands Using | 335 |
Index | 359 |
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affinity aliquots amino analysis antiphosphotyrosine antibodies approx assay bacterial beads BIAcore binding Bio-Rad Biochem Biol cDNA cellular centrifugation Chem chromatography cloning column concentration containing described in Subheading detection dilution EDTA electrophoresis elution enzyme ethanol expression filter fluorescence fraction fusion protein gene growth factor histidine hydrolysis immobilized immunoprecipitation Incubate inhibitor injection interaction isolated kinase activity LexA ligand lysis buffer MAPK medium membrane method mg/mL microcentrifuge Molecular NaCl nitrocellulose Note overnight pathway pellet peptide phage phosphoamino acid phosphopeptide phosphoprotein phosphorylation phosphotyrosine pipet plasmid plate prepared probe protein kinase Protocols purification radioactive reaction reagents receptor ECD receptor tyrosine kinases residues Resuspend room temperature sample screen SDS-PAGE Sepharose sequence SH2 domain Sigma signaling Smad sodium solution specific step stored at 4°C substrate supernatant TBST tion transfection transfer Tris-HCl trypsin tube vector volume wash Western blotting yeast µg/mL