An automated filtration assay for protein kinase C ligands.
[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; Kd's were 1.95 +/- 0.3 and 2.2 +/- 0.55 nM, 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.
Gross
J L
JL
Medical Products Department, E.I. du Pont de Nemours & Co., Inc., Wilmington, Delaware 19898.
Enders-Stephens
L
L
Herblin
W F
WF
D000818:Animals; D001322:Autoanalysis; D001667:Binding, Competitive; D001921:Brain / Q000201:enzymology; D005374:Filtration / Q000379:methods*; D008024:Ligands; D051379:Mice; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C / Q000378:metabolism*
D008024:Ligands; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C