An automated filtration assay for protein kinase C ligands

Abstract

[ 3H]Phorbol dibutyrate ([ 3H]PDBu) binding to soluble mouse brain protein kinase C (PKC) was established in a 96-well microtiter plate assay. [ 3H]PDBu-PKC receptor complexes were rapidly aspirated from wells, filtered, and washed onto glass fiber filter mats using an automated cell harvester. Results were compared to a modification of a previously described assay in which components were incubated in tubes, and manually delivered and washed onto filters with a manifold filtration apparatus. Both 96-well plate and tube assays gave qualitatively and quantitatively similar results since: (i) [ 3H]PDBu binding to PKC was phosphatidylserine (PS) dependent and calcium stimulatable; (ii) the amounts of [ 3H]PDBu bound by filters with each technique at receptors excess were similar, 3.2 ± 0.3 and 3.1 ± 0.4 pmol respectively; and (iii) the affinities of [ 3H]PDBu for PKC were comparable; K d's were 1.95 ± 0.3 and 2.2 ± 0.55 n m, respectively. The 96-well plate assay was more accurate and rapid than the tube assay. The microtiter plate assay was adapted for use with [ N,N-dimethyl- 3H] N,N-dimethylstaurosporine ([ 3H]DMS). With [ 3H]PDBu and [ 3H]DMS as ligands, the 96-well plate method was used for the rapid discrimination of agents which bound selectively at the regulatory and/or catalytic domains of PKC.