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 4° 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 4° C. Transfer supernatant to a new microcentrifuge or 15 ml conical centrifuge tube at 4° 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 4° C.
Add 20 µl of appropriate agarose conjugate suspension (Protein A-Agarose: sc-2001, Protein G PLUS-Agarose: sc-2002, Protein A/G PLUS-Agarose: sc-2003, or Protein L-Agarose: sc-2336). Cap tubes and incubate at 4° 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 4° C. Carefully aspirate and discard supernatant.
Wash pellet four times with 1.0 ml RIPA buffer (sc-24948) (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-280628)), 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 30° C. Place on ice.
Terminate the reaction by adding an equal volume of Electrophoresis Sample Buffer, 2X (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 -20° 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.
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