Rapid measurement of protein kinase and phosphatase activities by slot-filtration.
A slot-filtration method has been developed for the detection and quantitation of protein kinase and phosphatase activities. In this technique, after kinase-dependent phosphorylation or phosphatase-dependent dephosphorylation of different substrates, samples are transferred under vacuum onto nitrocellulose using a slot-blotting apparatus. Non-incorporated or released radioactivity is then removed by filtration and washing under vacuum. Quantitation is performed by scintillation or Cerenkov counting of the excised membrane slots. Application of the method to the assay of four different protein kinases (protein kinase N, cyclic AMP-dependent protein kinase and calcium/calmodulin-dependent protein kinases type I and type III) and one phosphatase is presented. A number of protein substrates with varying molecular masses and isoelectric points were found suitable for the slot-filtration technique. The method is applicable to impure as well as purified kinase and phosphatase preparations, can be used over a wide range of concentrations of substrates, has a very low background of nonspecific ATP binding and provides highly reproducible data. The slot-filtration method can also be adapted for use with ion-exchange paper, particularly for assays using peptides as substrates. The technique, with either nitrocellulose or ion-exchange paper, can be used to rapidly process large numbers of samples and can be simultaneously applied to direct comparison of different kinases, phosphatases and/or substrates in the same experiment.
Volonté
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C
Department of Pathology, Columbia University, New York, NY 10032.
Nichols
R A
RA
Greene
L A
LA
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002852:Chromatography, Ion Exchange; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel; D054736:Elongation Factor 2 Kinase; D005069:Evaluation Studies as Topic; D005374:Filtration / Q000379:methods*; D008567:Membranes, Artificial; D016716:PC12 Cells; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis*; D011494:Protein Kinases / Q000032:analysis*; D013997:Time Factors
D008567:Membranes, Artificial; D003101:Collodion; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D054736:Elongation Factor 2 Kinase; D010744:Phosphoric Monoester Hydrolases