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High Density Protein Kinase Assay With Subattomole SensitivityPromega Corporation, 2800 Woods Hollow Road, Madison, WI 53711-5399
A rapid assay system based on incorporation of [
Journal of Biomolecular Screening, Vol. 3, No. 1,
29-35 (1998) |
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-32P]ATP into biotinylated peptide substrates and their subsequent capture onto a high capacity streptavidin-coated membrane, SAM2TM, has been developed for the detection of protein kinases. The system uses prenumbered and partially cut membrane squares for analyzing a limited number of samples or can be formatted to analyze up to 1,536 samples per microtiter plate footprint of 7.0 cm X 10.6 cm. The high biotin-binding capacity and low background of the membrane allows the use of nearly saturating amounts of most substrates, giving this system very high signal-to-noise ratios at low enzyme concentrations. Using cAMP-dependent Protein Kinase A (PKA) as a model system, as little as 0.3 amol of purified enzyme in 0.2 µl can be detected with a linear response range of over 3 orders of magnitude. cAMP-dependent kinase activity can be measured directly in tissue extracts by using a specific substrate and harsh washing procedures to reduce nonspecific backgrounds from proteins phosphorylated by other kinases. For increased assay flexibility, results can be analyzed either by PhosphorImagerTM quantitation or by scintillation counting.