Immobilized Immunoprecipitation Protocol / (For Analysis By Western Immunoblotting)

A. Solutions and Reagents

NOTE: Prepare solutions with Milli-Q or equivalently purified water.

1. 1X Phosphate Buffered Saline (PBS)
2. 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*.

3. 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

1. Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
2. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold PBS.
3. 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.
4. Scrape cells off the plates and transfer to microcentrifuge tubes. Keep on ice.
5. Sonicate samples on ice four times for 5 seconds each.
6. Microcentrifuge for 10 minutes at 4°C, and transfer the supernatant to a new tube. If necessary, lysate can be stored at –80°C.

C. Immunoprecipitation

1. Take 200 μl cell lysate and add 10 µl of the immobilized antibody, incubate with gentle rocking overnight at 4°C.
2. Microcentrifuge for 30 seconds at 4°C. Wash pellet five times with 500 µl of 1X cell lysis buffer. Keep on ice during washes.
3. Resuspend the pellet with 20 μl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 seconds.
4. Heat the sample to 95–100°C for 2–5 minutes.
5. Load the sample (15–30 µl) on SDS-PAGE gel (12–15%).
6. Analyze sample by Western blotting (see Western Immunoblotting Protocol).