Immunoprecipitation General Protocol
The protocols described below are for general application. Any product specific protocol supercedes these general recommendations.
II. Immunoprecipitation Protocol
A. Lysis buffers
B. Preparation of lysates
C. Pre-clearing lysates
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.
II. Immunoprecipitation Protocol
A. 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.)
B. 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.
C. Pre-clearing the lysates
If using a monoclonal antibody choose protein G-coupled Sepharose beads; if using a polyclonal antibody, protein A-coupled Sepharose beads are usually suitable (refer to Table 1).
7. Transfer 50ul of the Protein G beads slurry to an eppendorf tube and add 450ul cold Lysis Buffer. Spin at 10,000xg for 30 seconds and remove the Lysis Buffer.
8. Wash one more time with 500ul of cold Lysis Buffer. Resuspend the beads in 50ul of cold Lysis Buffer.
9. Add the 50ul of prepared Protein G slurry and 500ul of Cell Lysate to an eppendorf tube and incubate on ice for 30-60 minutes.
10. Spin at 10,000xg for 10 minutes at 4°C and transfer the supernatant to a fresh eppendorf. If any bead has been transferred, spin again and carefully transfer the supernatant to another fresh eppendorf tube.
11. Add 5-10ug of antibody to the eppendorf tube containing the cold precleared lysate. Note: This concentration of monoclonal antibody is suggested as a starting point. Incubate at 4°C for 1 hour.
12. Add 50ul of washed Protein G slurry in prechilled Lysis Buffer (prepared as instructed in Preclearing Step 1 above).
13. Incubate for 1 hour at 4°C on a rocking platform or a rotator.
14. Spin the eppendorf tube at 10,000xg for 30 seconds at 4°C.
15. Carefully remove the supernatant completely and wash the beads 3-5 times with 500ul of Lysis Buffer. To minimize background, care should be given to remove the supernatant completely in these washes.
16. After the last wash, aspirate supernatant and add 50ul of 1X Laemmli sample buffer to bead pellet.
17. Vortex and heat to 90-100°C for 10 minutes.
18. Spin at 10,000xg for 5 minutes, collect supernatant and load onto the gel. Supernatant samples can be collected and kept frozen at this point if the gel is to be run later.
Table 1. Choosing the Correct Beads-Summary
|Species||Immunoglobulin Isotype||Protein A||Protein G|
|Human||IgM||Use anti Human IgM||Use anti Human IgM|
|Mouse||IgM||Use anti Mouse IgM||Use anti Mouse IgM|
|Guinea Pig||All isotypes||+++||++|
a. Sample possibly degraded by proteases. Include additional protease inhibitors in lysis and wash buffers. Keep sample cold at all times.
b. Antibody concentration too low. Increase concentration of precipitating antibody.
c. Precipitating antibody did not bind to Protein A/Protein G Agarose. Use Protein G/Protein A Agarose.
2. Problem: Nonspecific proteins bind to protein(G/A)-agarose or are trapped in the protein (G/A)-antibody-antigen immunocomplex.
a. Repeat the pre-clearing procedure several times before doing Immunoprecipitation.
b. Increase the washing time after immunoprecipitation.
c. Increase the stringency of the washes. For example, wash with a buffer containing a higher concentration of detergent or salt.
d. During binding and wash steps, let protein (G/A)-antibody-antigen immunocomplex settle by gravity rather than by centrifugation.