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.
- 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.
- 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.
- Protein A agarose Beads: (#9863) Add 5 ml of 1X PBS to 1.5 g of protein A agarose beads. Incubate for 2 hours at 4°C with gentle agitation; Pellet beads by centrifugation. Wash pellet twice with PBS. Resuspend beads in 1 volume of PBS.
- 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 denaturing conditions, remove media and rinse cells once with PBS.
- 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).
- Immediately scrape cells off the plate and transfer to 2.5 ml tubes.
- Boil for 10 minutes.
- 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.
- 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 5 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).
posted June 2005
revised January 2012