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: CST recommends adding 1 mM PMSF before use*.
- 3X SDS Sample Buffer: (#7722) 187.5 mM Tris-HCl (pH 6.8 at 25°C), 6% w/v SDS, 30% glycerol, 150 mM DTT, 0.03% w/v bromophenol blue
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 plus 1 mM PMSF* to each plate (10 cm) and incubate the plates on ice for 5 minutes.
- Scrape cells off the plates and transfer to microcentrifuge tubes. Keep on ice.
- Sonicate samples on ice four times for 5 seconds each.
- Microcentrifuge for 10 minutes at 4°C, and transfer the supernatant to a new tube. If necessary, lysate can be stored at –80°C.
- Take 200 μl cell lysate and add 10 µl of the immobilized antibody, incubate with gentle rocking overnight at 4°C.
- Microcentrifuge for 30 seconds at 4°C. Wash pellet five times with 500 µl of 1X cell lysis buffer. Keep on ice during washes.
- Resuspend the pellet 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 (12–15%).
- Analyze sample by Western blotting (see Western Immunoblotting Protocol).