Generation of phosphorylation state-specific SRC-class kinase antibodies for analysis of kinase activation.
Protein phosphorylation is a major molecular mechanism by which cellular function is regulated. In order to accomplish rapid and specific biochemical changes via phosphorylation, the activity of a protein kinase must be dynamically regulated. Historically, the activity of each protein kinase has been analyzed using a unique in vitro biochemical assay with a specific substrate and detection procedure. These assays require the use of radioactivity and are often labor intensive. Upon activation, most protein kinases autophosphorylate. Thus, a technical approach to detect changes in kinase activity is to measure autophosphorylation. The purpose of this protocol is to provide a detailed stepwise procedure for measuring the regulation of Src-class kinase activity using phosphorylation state-specific antibodies. Antibodies to a phosphorylated peptide derived from the autophosphorylation site of Src-family kinases are developed and affinity purified. The purified antibodies are used to analyze the regulation of Src and Fyn activity in a mouse muscle cell line. It is anticipated that the utility of these phosphorylation state-specific antibodies will ultimately result in the development of similar antibodies useful for analyzing the activity of many different kinases.
Miermont
A M
AM
EP08 Research Bld, Department of Neuroscience, Georgetown University Medical Center, 3970 Reservoir Rd NW, Washington, DC 20007-2197, USA.
Mohamed
A S
AS
Swope
S L
SL
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification; D000915:Antibody Affinity; D000918:Antibody Specificity; D002460:Cell Line; D002846:Chromatography, Affinity; D004337:Drug Carriers / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D051379:Mice; D009132:Muscles / Q000201:enzymology; D009842:Oligopeptides / Q000276:immunology / Q000378:metabolism; D010766:Phosphorylation; D011817:Rabbits; D012710:Serum Albumin, Bovine / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases / Q000276:immunology* / Q000378:metabolism*
D000906:Antibodies; D004337:Drug Carriers; D009842:Oligopeptides; D012710:Serum Albumin, Bovine; D014443:Tyrosine; D019061:src-Family Kinases