Troubleshooting  Immunoprecipitation

The most common challenge with immunoprecipitation is trying to lower the number and type of background proteins that contaminate the washed immune complexes. Background problems can arise from many different sources and can be either specific or nonspecific. The following are suggestions to deal with nonspecific background problems:

1. Preclear the lysates with Protein A/G agarose beads prior to adding the primary antibody.
2. Add saturating amounts of competitor proteins, such as BSA, gelatin, acetone powders, or blotto.
3. Spin the lysate at 100,000 x g for 30 min (discard pellet) prior to addition of primary antibody.
4. Centrifuge the antibody at 100,000 x g for 30 min (discard pellet) and titrate.
5. Increase the number of washes. “Soak” solid phase in the wash buffers for 10 min per wash.
6. Decrease primary antibody concentration.
7. Decrease primary antibody incubation time.
8. If using rabbit anti-mouse immunoglobulin (precipitating secondary antibody) in conjunction with a monoclonal antibody, check the background due to precipitating secondary antibody alone. Titrate if necessary.
9. Lower the number of counts per minute (cpm) of the radiolabel used to the minimum needed for antigen detection.
10. If specific proteins remain, remember that your antigen may consist of more than one polypeptide chain. Additional bands may also represent polypeptides that associate with the target antigen in vivo that have co-immunoprecipitated with the target antigen.
11. Run no primary and protein A only extract control lanes. This will aid in determining background bands arising from the Protein A beads or extract alone.
12. Multiple bands detected below the anticipated molecular weight may indicate proteolytic degradation of sample. Prepare new lysates with fresh protease inhibitors added.
13. Try a different antibody.