Immunoprecipitation Protocol / (For Denatured Proteins) (IP / Denatured Protein)

This protocol is intended for immunoprecipitation of denatured proteins where the epitope of interest may not be accessible in the native conformation.

A. Solutions and Reagents

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

1. Denaturing Cell Lysis Buffer: 50 mM Tris (pH 7.5), 70 mM β-Mercaptoethanol (β-ME) Add β-ME just prior to use. Pre-boil for 10 minutes.
2. Cell Lysis Buffer (1X): (#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.

3. Protein A or G Agarose Beads: Use Protein A (#9863) for rabbit IgG pull down and Protein G for mouse IgG pull down.
4. 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 denaturing conditions, remove media and rinse cells once with PBS.
3. Remove PBS and add 0.4 ml Denaturing Cell Lysis Buffer, (preboiled for 10 minutes immediately prior to use), to each plate (150 x 25 mm).
4. Immediately scrape cells off the plate and transfer to 2.5 ml tubes.
5. Boil for 10 minutes.
6. Add 4 volumes (1.6 ml) 1X ice-cold Cell Lysis Buffer. This is the cell lysate. If necessary, lysate can be stored at –80°C.

C. Immunoprecipitation

1. Add primary antibody to 200 µl cell lysate; incubate with gentle rocking overnight at 4°C.
2. Add either Protein A or G 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 5 times with 500 µl of 1X Cell Lysis Buffer. Keep on ice during washes.
4. Resuspend the pellet with 20 µl 3X SDS Sample Buffer. Vortex, then microcentrifuge for 30 seconds.
5. Heat the sample to 95–100°C for 2–5 minutes.
6. Load the sample (15–30 µl) on SDS-PAGE gel (12–15%).
7. Analyze sample by Western blotting (see Western Immunoblotting Protocol).