title: A semiautomated 96-well plate assay for protein kinase C.
abstract: 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.
authors.affiliation: Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510.
authors.forename: D T
authors.initials: DT
authors.lastname: Aftab
authors.forename: W N
authors.initials: WN
authors.lastname: Hait
mesh_terms: D000818:Animals; D001921:Brain / Q000201:enzymology; D008297:Male; D008722:Methods; D011493:Protein Kinase C / Q000032:analysis*; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D011493:Protein Kinase C