Solutions and Reagents

NOTE: Prepare solutions with Milli-Q or equivalently purified water.

  1. 1X Phosphate Buffered Saline (PBS) (#9808, 20X PBS)
  2. 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.
  3. Protein A or G Magnetic Beads: Use Protein A (#8687) for rabbit IgG pull down and Protein G (#8740) for mouse IgG pull down.
  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. (#7722, Blue Loading Buffer Pack)
  5. Magnetic Separation Rack (#7017)

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.

Immunoprecipitation

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).

  1. Take 200 μl cell lysate and add primary antibody. Incubate with gentle rocking overnight at 4°C.
  2. Vortex the stock tube briefly to resuspend the magnetic beads.
  3. 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.
  4. 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.
  5. 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.
  6. Resuspend the pellet with 20-40 μl 3X SDS sample buffer and vortex.
  7. Heat the sample to 95–100°C for 5 minutes and microcentrifuge for 1 minute at 14,000 X g.
  8. Load the sample (15–35 μl) on SDS-PAGE gel.
  9. 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 pre-washed Protein A or G magnetic beads (see step section C, steps 2 and 3).
  2. Incubate at room temperature for 10-30 minutes or at 4°C for 1-2 hours.
  3. 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.
  4. 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

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