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A Specific Immunoprecipitation Assay for the Protein Kinase FA/Glycogen Synthase Kinase 3

✍ Scribed by J. Vanlint; R.L. Khandelwal; W. Merlevede; J.R. Vandenheede

Publisher
Elsevier Science
Year
1993
Tongue
English
Weight
551 KB
Volume
208
Category
Article
ISSN
0003-2697

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✦ Synopsis

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.

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