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.

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.

3. 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.
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 nondenaturing conditions, remove media and rinse cells once with ice-cold PBS.
3. 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.
4. Scrape cells off the plates and transfer to microcentrifuge tubes. Keep on ice.
5. Sonicate samples on ice three times for 5 seconds each.
6. 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.

C. Immunoprecipitation

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).

1. Take 200 μl cell lysate and add primary antibody. 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 five 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 and microcentrifuge for 1 minute at 14,000 X g.
6. Load the sample (15–30 μl) on SDS-PAGE gel (12–15%).
7. Analyze sample by Western blotting (see Western Immunoblotting Protocol: Western BSA or Western Milk).

Cell Lysate Pre-Clearing (Optional)

1. Take 200 μl cell lysate and add to either Protein A or G agarose beads (20 μl of 50% bead slurry).
2. Incubate at 4°C for 30 – 60 minutes.
3. Spin for 10 minutes at 4°C. Transfer the supernatant to a fresh tube.
4. 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.