Development and validation of a competitive AKT serine/threonine kinase fluorescence polarization assay using a product-specific anti-phospho-serine antibody.
A competitive fluorescence polarization (FP) assay has been developed for the serine/threonine kinase, AKT. The FP assay has been formatted in a 384-well microtiter plate and automated using a pipeting workstation with performance suitable for high-throughput screening. The assay design utilizes a fluorescent phosphorylated peptide complexed to a product-specific anti-phospho-serine antibody. When unlabeled substrate is phosphorylated, by the kinase, the product competes with the fluorescent phosphorylated peptide for the antibody. The fluorescent phosphorylated peptide is then released from the antibody into solution resulting in a loss in polarization signal. Seven fluorescent phosphorylated peptides and 19 antibodies were evaluated for this assay. RARTSpSFAEPGK-Fl peptide and anti-phospho-GSK-3alpha Ser21 antibody gave the best affinity and change in polarization signal. The apparent kinetic constants were calculated for the FP assay and were consistent with reported values. The FP assay was validated with known inhibitors and the results compared to a radioactive Flashplate transfer assay, utilizing [(33)P]ATP and a biotinylated substrate, also developed in our laboratory. The IC(50) values generated were comparable between the two methods suggesting the competitive FP assay and Flashplate assay have similar sensitivities and abilities to identify inhibitors during screening.
Turek
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High Throughput Screening, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033-1300, USA. tammy.turek@spcorp.com
Small
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Bryant
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Hill
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D000906:Antibodies / Q000737:chemistry* / Q000276:immunology; D001667:Binding, Competitive; D005454:Fluorescence Polarization / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D007700:Kinetics; D010455:Peptides / Q000737:chemistry*; D010761:Phosphorus Radioisotopes / Q000737:chemistry; D010768:Phosphoserine / Q000276:immunology; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000037:antagonists & inhibitors; D011518:Proto-Oncogene Proteins / Q000032:analysis* / Q000037:antagonists & inhibitors*; D051057:Proto-Oncogene Proteins c-akt; D012680:Sensitivity and Specificity; D019311:Staurosporine / Q000378:metabolism / Q000494:pharmacology
D000906:Antibodies; D005456:Fluorescent Dyes; D010455:Peptides; D010761:Phosphorus Radioisotopes; D011518:Proto-Oncogene Proteins; D010768:Phosphoserine; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D019311:Staurosporine