Immunoprecipitation Protocol Utilizing Magnetic Separation / (For Analysis By Western Immunoblotting)
Solutions and Reagents
NOTE: Prepare solutions with Milli-Q or equivalently purified water.
- 1X Phosphate Buffered Saline (PBS) (#9808, 20X PBS)
- 1X Cell Lysis Buffer (#9803, 10X Cell Lysis Buffer): 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 Magnetic Beads: Use Protein A (#8687) for rabbit IgG pull down and Protein G (#8740) for mouse IgG pull down.
- 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. (#7722, Blue Loading Buffer Pack)
- Magnetic Separation Rack (#7017)
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 or G magnetic beads (See section below).
- Take 200 μl cell lysate and add primary antibody. Incubate with gentle rocking overnight at 4°C.
- Vortex the stock tube briefly to resuspend the magnetic beads.
- Pre-wash the protein A or G magnetic beads by adding 30-50 μl of bead slurry to a clean tube containing 500 μl 1X cell lysis buffer. Vortex and place the tube in a magnetic separation rack for 10-15 seconds. Once the solution is clear, carefully remove all of the supernatant.
- Transfer the lysate and antibody (immunocomplex) solution to the tube containing the washed magnetic bead pellet. Incubate with gentle rocking for 10-30 minutes at room temperature.
- Place the tubes containing the beads in the magnetic separation rack and wait 10-15 seconds for the solution to clear before carefully removing the supernatant. Wash pellet 3 times with 500 µl of 1X cell lysis buffer.
- Resuspend the pellet with 20-40 μl 3X SDS sample buffer and vortex.
- Heat the sample to 95–100°C for 5 minutes and microcentrifuge for 1 minute at 14,000 X g.
- Load the sample (15–35 μl) on SDS-PAGE gel.
- Analyze sample by Western blotting (see Western Immunoblotting Protocol: Western BSA or Western Milk).
Cell Lysate Pre-Clearing (Optional)
- Take 200 μl cell lysate and add to pre-washed Protein A or G magnetic beads (see step section C, steps 2 and 3).
- Incubate at room temperature for 10-30 minutes or at 4°C for 1-2 hours.
- Using a magnetic separation rack, separate the beads from the lysate, transfer the pre-cleared lysate to a clean tube and discard the magnetic bead pellet.
- Proceed to step 1 of Immunoprecipitation.
NOTE: For proteins with molecular weights in the range of around 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 interference produced by denatured heavy chains. For proteins with molecular weights in the range of around 25 kDa, Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb #3678 is recommended to minimize interference produced by denatured light chains.
posted December 2011