Immunoprecipitation of Denatured Proteins

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): 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: 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. (Can be stored for 2 weeks at 4°C.)
4. 3X SDS Sample Buffer: 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

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