Determination of specific protein kinase activities using phosphorus-33.
The immune complex kinase assay is the most widely applied method to assess the catalytic activity of protein tyrosine kinases. It offers the advantage that the activity of a single selected enzyme can be determined, and that the enzyme activity can be normalized for the amount of enzyme in a parallel immunoblotting experiment. Here, we describe the use of the recently introduced isotope phosphorus-33 for the protein kinase assay. The lower energy of 33P, compared with the traditionally applied 32P, allows the simultaneous examination of the amount of enzyme with 125I-labeled antibodies. By analysing one and the same sample for both kinase activity and protein amount, the variation between parallel processed samples is avoided. Using this method, specific kinase activities can be calculated with high precision. The assay is particularly useful for the detection of cytokine and growth factor-induced activation of kinases, as changes in enzyme amounts by subcellular relocalization can be distinguished.
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Department of Hematology, University Hospital Utrecht, Netherlands.
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D002277:Carcinoma / Q000201:enzymology; D005909:Glioblastoma / Q000201:enzymology; D006801:Humans; D015151:Immunoblotting; D008722:Methods; D009363:Neoplasm Proteins / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D019000:Phosphotyrosine / Q000378:metabolism; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D012680:Sensitivity and Specificity; D012878:Skin Neoplasms / Q000201:enzymology; D014407:Tumor Cells, Cultured
D009363:Neoplasm Proteins; D010761:Phosphorus Radioisotopes; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases