High Throughput Screening Protein Kinase Assays Optimized for Reaction, Binding, and Detection Totally within a 96-Well Plate

Abstract

Signal transduction assays, particularly for protein kinases, are an area of increasing interest and activity for laboratories investigating the regulation of cellular functions. The traditional kinase assay methods require the tedious and time consuming manipulation of phosphocellulose disks typically used to bind the phosphorylated substrate. Drug discovery research requires the availability of rapid and reliable procedures to evaluate large numbers of samples for bioactivity. The 96-well phosphocellulose MultiScreen(r) assay plates were specifically developed to meet these assay requirements. A cyclic-AMP-dependent protein kinase A (PKA) assay was optimized to be performed entirely within the phosphocellulose MultiScreen plate, including reagent and sample addition, incubation, washing, and direct microplate scintillation counting. The protocol is directly adaptable to a high throughput kinase screen. Both the Kemptide peptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) and the histone H1 protein were used as the phosphorylation substrates. Crude and purified PKA enzymes were found to have a sensitivity of 0.4 U for Kemptide substrate, which was comparable to the assay performed by the traditional transfer to phosphocellulose paper. The results demonstrate that kinase assays can be performed entirely in a MultiScreen phosphocellulose plate.