Immunoprecipitation Protocol (For Analysis By Kinase Assay)

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: Add 1 mM PMSF immediately prior to use.

1. 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.
2. 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

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 to each plate (10 cm) and incubate the plate on ice for 5 minutes.
4. Scrape cells off the plate and transfer to microcentrifuge tubes. Keep on ice.
5. Sonicate on ice three times for 5 seconds each.
6. 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.

C. Immunoprecipitation

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.

1. Add primary antibody to 200 μl cell lysate; incubate with gentle rocking overnight at 4°C.
2. Add protein A agarose beads (20 μl of 50% bead slurry). Incubate with gentle rocking for 1–3 hours at 4°C.
3. Microcentrifuge for 30 seconds at 4°C. Wash pellet two times with 500 μl of 1X cell lysis buffer. Keep on ice during washes.
4. Wash pellet twice with 500 μl 1X kinase buffer. Keep on ice.

D. Kinase Assay

1. Suspend pellet in 40 μl 1X kinase buffer supplemented with 200 μM ATP and substrate.
2. Incubate 30 minutes at 30°C.
3. Terminate reaction 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.
6. Analyze sample by Western blotting (see Western Immunoblotting Protocol).