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) (#9808)
2. 1X Cell Lysis Buffer: (#9803) To prepare 10 ml of 1X cell lysis buffer, add 1 ml 10X cell lysis buffer to 9 ml dH₂O, mix.

   NOTE: Add 1 mM PMSF (#8553) immediately prior to use.

3. Protein A or G Agarose Beads: Use Protein A (#73778) for rabbit pull down and Protein G (#70024) for mouse IgG pull down.
4. 3X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer.
5. Magnetic Separation Rack: (#7017) or (#14654)

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

Cell Lysate Pre-Clearing (Highly Recommended)

A cell lysate pre-clearing step is highly recommended to reduce non-specific protein binding to the Protein A or G Magnetic beads. Pre-clear enough lysate for test samples and isotype controls.

1. Briefly vortex the stock tube to resuspend the magnetic beads.

   IMPORTANT: Pre-wash #73778 and #70024 magnetic beads just prior to use:

2. Transfer 20 μl of bead slurry to a clean tube. Place the tube in a magnetic separation rack for 10-15 seconds.

   Carefully remove the buffer once the solution is clear. Add 500 μl of 1X cell lysis buffer to the magnetic bead pellet, briefly vortex to wash the beads. Place tube back in magnetic separation rack. Remove buffer once solution is clear. Repeat washing step once more.

3. Add 200 μl cell lysate to 20 μl of pre-washed magnetic beads.

   IMPORTANT: The optimal lysate concentration will depend on the expression level of the protein of interest. A starting concentration between 250 μg/ml - 1.0 mg/ml is recommended.

4. Incubate with rotation for 20 minutes at room temperature.
5. Separate the beads from the lysate using a magnetic separation rack, transfer the pre-cleared lysate to a clean tube, and discard the magnetic bead pellet.
6. Proceed to immunoprecipitation section.

Immunoprecipitation

IMPORTANT: Appropriate isotype controls are highly recommended in order to show specific binding in your primary antibody immunoprecipitation. Use Normal Rabbit IgG #2729 for rabbit polyclonal primary antibodies, Rabbit (DA1E) mAb IgG XP^® Isotype Control #3900 for rabbit monoclonal primary antibodies, Mouse (G3A1) mAb IgG1 Isotype Control #5415 for mouse monoclonal IgG1 primary antibodies, Mouse (E5Y6Q) mAb IgG2a Isotype Control #61656 for mouse monoclonal IgG2a primary antibodies, Mouse (E7Q5L) mAb IgG2b Isotype Control #53484 for mouse monoclonal IgG2b primary antibodies, and Mouse (E1D5H) mAb IgG3 Isotype Control #37988 for mouse monoclonal IgG3 primary antibodies. Isotype controls should be concentration matched and run alongside the primary antibody samples.

1. Add primary antibody (at the appropriate dilution as recommended in the product datasheet) to 200 μl cell lysate. Incubate with rotation overnight at 4°C to form the immunocomplex.
2. Pre-wash magnetic beads (see Cell Lysate Pre-Clearing section, steps 1 and 2).
3. Transfer the lysate and antibody (immunocomplex) solution to the tube containing the pre-washed magnetic bead pellet.
4. Incubate with rotation for 20 minutes at room temperature.
5. Pellet beads using magnetic separation rack. Wash pellet five times with 500 μl of 1X cell lysis buffer. Keep on ice between washes.
6. Resuspend the pellet with 20-40 μl 3X SDS sample buffer, briefly vortex to mix, and briefly microcentrifuge to pellet the sample.
7. Heat the sample to 95-100°C for 5 min.
8. Pellet beads using magnetic separation rack. Transfer the supernatant to a new tube. The supernatant is the sample.
9. Load the sample (15-35 μl) on SDS-PAGE gel.
10. Analyze sample by Western blotting (see Western Immunoblotting Protocol: Western BSA or Western Milk ).

NOTE: For proteins with molecular weights of 50 kDa, we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (#45262) 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 in the range of around 25 kDa, Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) (#5127) is recommended to minimize interference produced by denatured light chains.