Roskoski-Assay

Protein kinase activity may be measured by using a modified radioactive assay [1].
The assay is performed in a mixture containing 50mM MOPS, pH 7.0, 10mM MgCI₂, 0.25mg/ml BSA, 100µM kemptide (peptide substrate), 100µM unlabeled ATP mixed with [γ-³²p] ATP (500-1000cpm/pmol) in a final volume of 50µI. Reaction is started by addition of the Cα subunit from activated PKA holoenzyme, type IIα and can be stopped after a 5 min. incubation at 30°C by spotting the reaction mix onto Whatman^® P-81 filters and soaking the filters four times in 75mM phosphoric acid (10ml per sample) for at least 5 min. After four washing steps rinse filters with ethanol, dry and count.
For the detection of phosphorylation in substrate proteins the phosphotransferase reaction can alternatively be stopped by taking aliquots of the mixture and adding SDS sample buffer. The phosphorylation status of the substrate proteins can subsequently be analysed using SDS PAGE and autoradiography [2].