Author: Bart's Cookbook This specific procedure was developed to assay the activity of the Lck kinase expressed from a retroviral vector in rat fibroblasts. You can obviously use fewer or more cells, depending on your needs. Don't skimp too much on the amount of S. aureus bugs that you use for precipitation or you will risk losses. 1. Immunoprecipitate the protein from 3 x 106 cells using 45 microliters S. aureus bugs. Resuspend the immunoprecipitates in 12 microliters kinase buffer (40 mM PIPES, pH 7.0, 10 mM MnCl2) at 4°C. - The kinase reaction is carried out at room temperature in a total volume of 20 microliters that contains: - 15 microCi gamma-32P-ATP - 5 microliters immunoprecipitate - 2 mMolar [Val5]-angiotensin II - Remove 5 microliter aliquots of the reaction mixture after 1, 3, and 5 minutes of reaction time. The incorporation of ATP is linear during this period. - The 5 microliter aliquots that are removed at each time point are mixed immediately with 15 microliters of 5% trichloroacetic acid (TCA). Let the samples sit on ice for at least 15 min to precipitate proteins. - Spin the samples in a microcentrifuge for 5 min at 4°C. - Spot 10 microliters of each supernatant onto a 2 x 2 cm phosphocellulose paper (Whatman p81). Wash the paper in 0.425% phosphoric acid (add 5 ml 85% phosphoric acid to 1 L H2O to make 0.425% phosphoric acid.) for 3 min (15 milliliters/piece of paper). Repeat the wash twice. Unincorporated ATP will be washed off the paper. - Add the pieces of paper to 5 ml scintillation fluid to and count. - Val5-angiotensin II is obtained from Sigma and stored as a 50 mM stock at -70°C. Note: If you get more than 100,000 cpm incorporated, you may be pegging the assay. In this case, repeat the experiment and use less of the precipitate in the assay. Consumption of most of the ATP can occur if you are studying proteins over-expressed by transient transfection.