Detection of protein kinase activity in sodium dodecyl sulfate-polyacrylamide gels.
A procedure is described for identifying protein kinase activity in protein samples following electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Protein kinase activity is detected by renaturation of the enzymes within the gel followed by phosphorylation with [gamma-32P]ATP of either substrates included in the polyacrylamide gel or of the kinase itself. Then, after removal of the unreacted [gamma-32P]ATP by washing the gel in the presence of an anion-exchange resin, the positions (Mr) of the protein kinase activity are visualized by autoradiography. Studies using a purified catalytic subunit of cAMP-dependent protein kinase indicate that enzyme concentrations as low as 0.01 microgram can easily be detected on gels containing 1 mg/ml casein. The technique is also useful for identifying active subunits of multisubunit enzymes. The active subunit of casein kinase II, for example, can readily be determined by renaturing the dissociated enzyme in gels containing casein. Putative protein kinases present in crude mixtures of proteins can also be detected following separation by gel electrophoresis and can be characterized on the basis of molecular weight and identity of the phosphorylated amino acid. Using this technique, at least three major protein kinases were detected in a mixture of proteins prepared by subfraction of red blood cell membranes.
Geahlen
R L
RL
Anostario
M
M
Low
P S
PS
Harrison
M L
ML
D000818:Animals; D047388:Casein Kinases; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004910:Erythrocyte Membrane / Q000201:enzymology; D008970:Molecular Weight; D010766:Phosphorylation; D011487:Protein Conformation; D011489:Protein Denaturation; D011494:Protein Kinases / Q000032:analysis*; D012967:Sodium Dodecyl Sulfate
D012967:Sodium Dodecyl Sulfate; D011494:Protein Kinases; D047388:Casein Kinases