A rapid assay system based on incorporation of [γ-32P]ATP into biotinylated peptide substrates and their subsequent capture onto a high capacity streptavidin-coated membrane, SAM2™, 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 PhosphorImager™ quantitation or by scintillation counting.
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