Analysis of tyrosine kinase activity in cell extracts using nondenaturing polyacrylamide gel electrophoresis.
A general procedure for detecting tyrosine kinase activity in crude or purified preparations using nondenaturing gel electrophoresis is presented. Samples are resolved by electrophoresis in minigels which are then incubated in an assay mixture containing [gamma-32P]ATP, poly(glutamic acid, tyrosine)4:1, and cofactors. Subsequently, gels are fixed and washed in trichloroacetic acid-pyrophosphate, dried, and analyzed by autoradiography or liquid scintillation counting. The procedure is simple and fast and allows analysis of different molecular weight forms of tyrosine kinase under linear kinetics at 37 degrees C without interference from phosphatases and proteases.
D000255:Adenosine Triphosphate / Q000378:metabolism; D002460:Cell Line; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D006098:Granulocytes / Q000201:enzymology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D007951:Leukemia, Myeloid / Q000201:enzymology; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000032:analysis* / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D019000:Phosphotyrosine; C027606:L-glutamic acid-L-tyrosine copolymer; D014443:Tyrosine; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases