A. Solutions and Reagents
NOTE: Prepare solutions with Milli-Q or equivalently purified water.
- 1X Phosphate Buffered Saline (PBS)
- 1X Cell Lysis Buffer: (#9803) 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml Leupeptin
NOTE: Add 1 mM PMSF immediately prior to use.
- Protein A Agarose Beads: (#9863) Add 5 ml of 1X PBS to 1.5 g of protein A agarose beads. Agitate for 2 hours at 4°C; pellet by centrifugation at 14,000 X g for 1 minute. Wash pellet twice with PBS. Resuspend beads in 1 volume of PBS.
- 1X Kinase Buffer: 25 mM Tris (pH 7.5), 5 mM β-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, 10 mM MgCl2
B. Preparing Cell Lysates
- Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
- To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold PBS.
- Remove PBS and add 0.5 ml 1X ice-cold cell lysis buffer to each plate (10 cm) and incubate the plate on ice for 5 minutes.
- Scrape cells off the plate and transfer to microcentrifuge tubes. Keep on ice.
- Sonicate on ice three times for 5 seconds each.
- Microcentrifuge for 10 minutes at 4°C, 14,000 X g and transfer the supernatant to a new tube. The supernatant is the cell lysate. If necessary, lysate can be stored at –80°C.
NOTE: For immunoprecipitations with immobilized primary antibody: Add 20 μl of immobilized antibody bead slurry to 200 μl cell lysate. Incubate with gentle rocking overnight at 4°C. Proceed to washing step 3.
- Add primary antibody to 200 μl cell lysate; incubate with gentle rocking overnight at 4°C.
- Add protein A agarose beads (20 μl of 50% bead slurry). Incubate with gentle rocking for 1–3 hours at 4°C.
- Microcentrifuge for 30 seconds at 4°C. Wash pellet two times with 500 μl of 1X cell lysis buffer. Keep on ice during washes.
- Wash pellet twice with 500 μl 1X kinase buffer. Keep on ice.
D. Kinase Assay
- Suspend pellet in 40 μl 1X kinase buffer supplemented with 200 μM ATP and substrate.
- Incubate 30 minutes at 30°C.
- Terminate reaction with 20 μl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 seconds.
- Heat the sample to 95–100°C for 2–5 minutes.
- Load the sample (15–30 μl) on SDS-PAGE gel.
- Analyze sample by Western blotting (see Western Immunoblotting Protocol).
posted June 2005
revised January 2012