A specific immunoprecipitation assay for the protein kinase FA/glycogen synthase kinase 3.
A specific immunoprecipitation assay has been developed for the accurate determination of the kinase FA/GSK3 activity in crude cell preparations. The assay is based on the production and isolation of polyclonal peptide antibodies toward the C-terminus of the rat GSK3-alpha and GSK3-beta isoforms. The respective forms of the kinases are captured as active immunocomplexes with immobilized secondary antibodies and their kinase activity toward the synthetic peptide P-GS1 can be accurately measured. Immunoprecipitation with a mixture of the two peptide antibodies allows for the detection and estimation of the total kinase FA/GSK3 activity in cellular extracts, whereas the alpha-peptide antibody specifically detects and measures the GSK3-alpha isoform. The contribution of GSK3-beta in the total kinase FA/GSK3 activity of cell fractions can be calculated.
Van Lint
J
J
Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit te Leuven, Belgium.
Khandelwal
R L
RL
Merlevede
W
W
Vandenheede
J R
JR
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D005069:Evaluation Studies as Topic; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D011233:Precipitin Tests / Q000379:methods*; D011494:Protein Kinases / Q000032:analysis* / Q000276:immunology; D051381:Rats; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured / Q000201:enzymology
D010455:Peptides; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases