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| | Protein Kinase Assays |
| Description: | Classical and novel PKC isotypes were assayed by scintillation proximity assay technology. In brief, the assay was performed in reaction buffer by incubating peptide substrate, [33P]ATP/ATP (10 uM), and PKC at a protein concentration varying from 25 to 400 ng/ml, and lipid vesicles containing 30 mol% phosphatidylserine, 5 mol% diacylglycerol (DAG), and 65 mol% phosphatidylcholine at a final lipid concentration of 0.5 uM. Incubation was performed for 60 min at room temperature. The reaction was stopped by adding streptavidin-coated scintillation proximity assay beads (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK) in PBS without Ca2+ and Mg2+. Incorporated radioactivity was measured in a MicroBetaTrilux counter (PerkinElmer, Schwerzenbach, Switzerland) for 1 min. |
| Affinity data for this assay | |
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