Protein kinase assay by paper-trichloroacetic acid method: high performance using phosphocellulose paper and washing an ensemble of samples on flat sheets.
Phosphocellulose paper has been found to be the paper of choice in assaying protein kinase activities using [gamma-32P]ATP by the trichloroacetic acid method of precipitation and washing. A study of binding of ATP of increasing concentrations at constant specific activity with Whatman 3MM or ATP-coated Whatman 3 MM papers (in vogue) versus phosphocellulose paper (proposed here) has shown that the latter has the least affinity for ATP when washing is done with either trichloracetic acid or trichloroacetic acid containing pyrophosphate. In an experiment where the placental cytosolic protein kinase was serially diluted, the phosphocellulose paper was found to give higher signal/noise ratios at all dilutions studied compared to the other two papers. With regard to the technological side of washing the papers, we have found that the traditional method of cutting papers into small squares before loading the samples is perhaps not the best. Instead, we propose the use of a flat sheet matrix for loading the samples because this method ensures uniformity of washing among the samples while shaking is performed on a simple shaker. In addition, the whole paper matrix can provide an almost instantaneous autoradiogram of hundreds of samples facilitating biochemical experimentation with protein kinases.
Beckman Research Institute of the City of Hope, Department of Molecular Genetics, Duarte, California 91010-0269.
D
D
Sahal
Y
Y
Fujita-Yamaguchi
D000255:Adenosine Triphosphate / Q000378:metabolism; D001345:Autoradiography; D005374:Filtration / Q000379:methods*; D006801:Humans; D010920:Placenta / Q000201:enzymology; D011494:Protein Kinases / Q000302:isolation & purification*; D014238:Trichloroacetic Acid
D014238:Trichloroacetic Acid; D000255:Adenosine Triphosphate; D011494:Protein Kinases