A semiautomated 96-well plate assay for protein kinase C

We have devised a rapid procedure for the assay of protein kinase C. Reactions (100 microliters) were set up in the wells of a 96-well assay plate and initiated one column at a time by the addition of [gamma-32P]ATP with an eight-channel pipettor. After incubation for 5 min at 30 degrees C, the reactions were terminated by the addition of 100 microliters of 25% trichloroacetic acid. The reaction mixtures were then filtered using a semiautomatic cell harvester, and transferred to scintillation vials using a filter punch apparatus. Direct comparison of this method to traditional techniques revealed a three- to fivefold increase in efficiency with equal sensitivity. This method is suitable for screening large numbers of inhibitors and activators of protein kinase C and appears to be applicable to other enzymes such as calmodulin kinases.