Immunoprecipitation Protocol / (For Analysis By Western Immunoblotting)
For shorter assay times please try our Immunoprecipitation Protocol Utilizing Magnetic Separation / (For Analysis By Western Immunoblotting).
A. Solutions and Reagents
NOTE: Prepare solutions with Milli-Q or equivalently purified water.
- 1X Phosphate Buffered Saline (PBS)
- 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.
- Protein A or G Agarose Beads: (Protein A #9863) Please prepare according to manufacturer’s instructions. Use Protein A for rabbit IgG pull down and Protein G for mouse IgG pull down.
- 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 nondenaturing conditions, remove media and rinse cells once with ice-cold PBS.
- Remove PBS and add 0.5 ml ice-cold 1X cell lysis buffer to each plate (10 cm) and incubate the plates on ice for 5 minutes.
- Scrape cells off the plates and transfer to microcentrifuge tubes. Keep on ice.
- Sonicate samples on ice three times for 5 seconds each.
- Microcentrifuge for 10 minutes at 14,000 X g, 4°C, and transfer the supernatant to a new tube. If necessary, lysate can be stored at –80°C.
Optional: It may be necessary to perform a lysate pre-clearing step to reduce non-specific binding to the Protein A/G agarose beads (See section below).
- Take 200 μl cell lysate and add primary antibody. Incubate with gentle rocking overnight at 4°C.
- 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.
- Microcentrifuge for 30 seconds at 4°C. Wash pellet five 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 and microcentrifuge for 1 minute at 14,000 X g.
- Load the sample (15–30 μl) on SDS-PAGE gel (12–15%).
- Analyze sample by Western blotting (see Western Immunoblotting Protocol: Western BSA, Western Milk).
Cell Lysate Pre-Clearing (Optional)
- Take 200 μl cell lysate and add to either Protein A or G agarose beads (20 μl of 50% bead slurry).
- Incubate at 4°C for 30 – 60 minutes.
- Spin for 10 minutes at 4°C. Transfer the supernatant to a fresh tube.
- Proceed to step 1 of Immunoprecipitation.
NOTE: For proteins with molecular weights of 50 kDa, we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (L57A3) mAb #3677 or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb #3678 as a secondary antibody to minimize masking produced by denatured heavy chains. For proteins with molecular weights of 25 kDa, Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb #3678 is recommended.
posted November 2006
revised January 2012