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.