Troubleshooting | Immunoprecipitation
Follow our immunoprecipitation troubleshooting guide to quickly identify and get solutions for common protocol issues.
First identify the problem with your immunohistochemistry staining from the options below:
High Background
Proteins are not detergent soluble:
- Remove supernatant immediately after centrifugations.
Incomplete washing:
- Wash well.
Non-specific proteins are binding:
- Beads are not pre-blocked enough with BSA. Make sure the BSA is fresh, and incubate fresh beads for 1 hour with 1% BSA. Wash 3 to 4 times in PBS before using.
- Try reducing the amount of sample loaded onto the beads.
Antibody Issues:
- Antibodies used are not specific enough – use an affinity purified antibody.
- Too much antibody- leads to non-specific binding. Check the recommended amount on the suppliers datasheet/ website.
Too many cells/too much protein in lysate leading to a lot of non-specific proteins in eluate:
- Reduce the number of cells/lysate used.
Antigen degrading during immunoprecipitation:
- Ensure fresh protease inhibitors are added when sample is lysed.
No eluted target protein detected
Target protein not expressed in sample used/Low level of target protein expression in sample used:
- Check the expression profile of the target protein to ensure it will be expressed in the cells of your samples. If there is low level of target protein expression, increase the amount of lysate used.
Insufficient antibody for capture of the target protein:
- Check that the recommended amount of antibody is being used.
Target protein has not eluted from the beads:
- Ensure you are using the correct elution buffer and that it is at the correct strength and pH for elution of the protein.
Antibody has not bound to immunosorbent beads:
- Ensure you are using the correct beads for the antibody isotype used.
High amount of antibody eluting
- Try reducing the amount of antibody. Cross-linking the antibody to the beads before the immunoprecipitation and eluting using a gentle glycine buffer gradient should significantly reduce the amount of antibody eluted.