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• Immunoprecipitation General Protocol

Immunoprecipitation General Protocol

The protocols described below are for general application. Any product specific protocol supercedes these general recommendations.

Background

Immunoprecipitation is a method that enables the purification of a protein. Additionally, one can use immunoprecipitation to confirm the identity or study biochemical characteristics, post-translational modifications, and expression levels of a protein of interest. An antibody for the protein of interest is incubated with a cell extract so that the antibody will bind the protein in solution. The antibody/antigen complex will then be pulled out of the sample using protein A/G-coupled agarose beads. This physically isolates the protein of interest from the rest of the sample. The sample can then be separated by SDS-PAGE for Western blot analysis.

Lysis Buffers

The ideal lysis buffer will leave proteins in their native conformation, minimizing denaturation of antibody binding sites while at the same time releasing adequate amounts of protein from the sample for subsequent analysis. Other variables that can affect the success of IP include salt concentration, divalent cation concentration, and pH.

1. NP-40 Cell Lysis Buffer: 50mM Tris-HCl pH 8.0, 150mM NaCl, 1% NP-40
2. Protease Inhibitor Cocktail (100X): PMSF, 5mg (50ug/ml), Aprotinin, 100ug (1μg/ml), Leupeptin, 100ug (1ug/ml), Pepstatin, 100ug (1ug/ml), 100% Ethanol bring up to 1ml, aliquot and keep at -20°C. (Add to Lysis buffer just before use.)
3. RIPA (RadioImmunoPrecipitation Assay) buffer (alternate) RIPA buffer is particularly useful for nuclear membrane disruption for nuclear extracts. RIPA buffer gives low background but can denature kinases. 50mM Tris HCl, pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS (10% sodium deoxycholate stock solution (5g into 50ml) must be protected from light.)

Preparation of Lysates

1. Harvest approximately 10e7 cells. Note: The total number of cells/ml and the cell equivalent loaded per lane of gel should be optimized specifically for each protein and antibody.
2. Wash cells with ~10 ml of PBS in a conical tube and spin at 400xg for 10 minutes. Discard supernatant.
3. Wash cells with ~10 ml of PBS in a conical tube and spin at 400xg for 10 minutes.
4. After the second wash, remove supernatant completely and resuspend the cell pellet in 1ml of cold Lysis Buffer containing 1X Protease Inhibitor Cocktail (final concentration of 10e7 cells/ml). Gently vortex the tube. Note: To effectively prevent protease action, the Lysis Buffer should be pre-chilled and all the following steps should performed at 4°C. For adherent monolayers, add 1ml of cold Lysis Buffer containing 1X Protease Inhibitor Cocktail per 100mm culture dish.
5. Place the tube or the dish on ice for 30 minutes, with occasional mixing. Spin cell lysate at 10,000xg for 15 minutes at 4°C.
6. Carefully collect supernatant, without disturbing the pellet, and transfer to a clean tube. The cell lysate can be frozen at this point for long-term storage at -70°C. Discard the pellet.