| • |
Remove medium from 100 mm cell culture plate (80–90% confluent monolayer) and wash once with PBS (Buffers and General Solutions). |
| • |
Add 1–3 ml ice cold RIPA buffer (sc-24948) to cell monolayer and incubate at 4º C for 10 minutes. (NOTE: the use of RIPA buffer may not be optimal for some kinases. Composition of lysis buffer may need to be optimized to maintain active kinase.) |
| • |
Disrupt cells by repeated passage through a 21-gauge needle and transfer to microcentrifuge or 15 ml conical centrifuge tube. |
| • |
Wash cell culture plate with addition of 1.0 ml ice cold RIPA buffer, 0.5% Triton X-100 (Triton X-100: sc-29112) and combine with original extract. |
| • |
Pellet cellular debris at 10,000xg for 10 minutes at 4º C. Transfer supernatant to a new microcentrifuge or 15 ml conical centrifuge tube at 4º C. |
| • |
Transfer 1.0 ml cell extract (supernatant from above step) to a 1.5 ml microcentrifuge tube. Add 1–10 µl (i.e., 0.2–2 µg) primary antibody (optimal antibody concentration should be determined by titration) and incubate for 1 hour at 4º C. |
| • |
Add 20 µl of appropriate agarose conjugate suspension (Protein A-Agarose, Protein G-Agarose, Protein A/G-Agarose, or Protein L-Agarose). Cap tubes and incubate at 4º C on a rocker platform or rotating device for 1 hour to overnight. |
| • |
Collect immunoprecipitates by centrifugation at 2,500 rpm (approximately 1,000xg) for 5 minutes at 4º C. Carefully aspirate and discard supernatant. |
| • |
Wash pellet four times with 1.0 ml RIPA buffer (more stringent) or PBS (Buffers and General Solutions) (less stringent), each time repeating centrifugation step above. |
| • |
Suspend pellet in 20 µl of the appropriate protein kinase assay buffer (e.g., 50 mM HEPES (HEPES: sc-29097), 0.1 mM EDTA (EDTA: sc-29092), 0.01% BRIJ® 35 (BRIJ® 35: sc-29087)), 0.1 mg/ml BSA, 0.1% β-mercaptoethanol, 0.15 M NaCl. Buffer composition will depend upon the kinase under study. |
| • |
Add 10–1000 ng peptide substrate. Peptide substrate concentration should be determined empirically for the substrate/enzyme/cell line used. |
| • |
Prepare 1 ml ATP mix: 930 µl appropriate protein kinase assay buffer, 6 µl 50 mM ATP, pH 7.0, 20 µl 2.0 M MgCl2, and 44 µl [γ32P]-ATP [10 mCi/ml]. Add 10 µl ATP mix per sample and incubate for 20 minutes at 30º C. Place on ice. |
| • |
Terminate the reaction by adding an equal volume of 2x electrophoresis sample buffer (sc-24945) and boil samples for 2–3 minutes. After boiling, samples may be centrifuged to pellet the agarose beads (optional); the supernatant is analyzed. Analyze samples by SDS-PAGE and autoradiography. Unused samples may be stored at -20º C. Alternatively, labeled peptides can be separated from unicorporated label by acid precipitation followed by collection on a filter and radioactivity determined by scintillation counting. Researchers may also choose to analyze immobilized peptides prepared by standard methods or offered commercially. |
