Rapid filtration assays for protein kinase C activity and phorbol ester binding using multiwell plates with fitted filtration discs.
In the conventional approach protein kinase activity and phorbol ester binding associated with protein kinase C (PKC) are measured by initially incubating samples in either test tubes or multiwell plates, followed by filtration of the terminated reaction mixture using either a manifold filtration device or a cell harvester. Here we report a method in which both the incubations and filtrations necessary for the determination of either protein kinase activity or phorbol ester binding are carried out in the same multiwell plate with fitted filtration discs made of polyvinylidene difluoride (Durapore membrane). Due to the very low binding of protein to these filters, there is no interference caused by these filters during the incubation period of the assays. The drawback with these filters compared to commonly used cellulose acetate membrane filters is that they retain less of the phosphate acceptor substrate histone H1 (only 15%) if filtered and washed with standard 5% trichloroacetic acid. However, this can be overcome by increasing the trichloroacetic acid concentration to 25% during filtration. For phorbol ester binding determinations, the samples are incubated with [3H]phorbol 12,13-dibutyrate in the microwells, the ligand bound PKC is adsorbed onto DEAE-Sephadex beads, and the beads then are filtered and washed in the same microwells. Furthermore, this multiwell filtration approach can also be adopted to previously described cytosolic phorbol ester receptor assays, which have the broader conditions for optimal binding to receptors. Durapore membrane filters are found to work well for punching into scintillation vials and there is complete recovery of the radioactivity retained with the filters. In the protein kinase assay the background radioactivity is very low (< 200 cpm) and in the phorbol ester binding assay the nonspecific binding is less than 1%. Thus, these low background values result in at least a fourfold increase in sensitivity for these assays. Since the incubations and filtrations are carried out in the same well without any transfer of the sample, the coefficient of variation in multiple determinations is found to be low. Furthermore, this method is rapid and more convenient for analyzing a larger number of samples than conventional methods which use test tubes, and it is less expensive to set up compared to the automated methods that use a cell harvester.
Gopalakrishna
R
R
Department of Pharmacology and Nutrition, USC School of Medicine, Los Angeles 90033.
Chen
Z H
ZH
Gundimeda
U
U
Wilson
J C
JC
Anderson
W B
WB
D000818:Animals; D003600:Cytosol / Q000201:enzymology; D005374:Filtration / Q000295:instrumentation; D006657:Histones / Q000302:isolation & purification; D008832:Microchemistry / Q000295:instrumentation; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism*; D011145:Polyvinyls; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D012015:Reference Standards; D012680:Sensitivity and Specificity; D005465:Fluorocarbon Polymers
D006657:Histones; D015240:Phorbol 12,13-Dibutyrate; D011145:Polyvinyls; D011493:Protein Kinase C; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers