troubleshooting of Co-IP

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Co-immunoprecipitation,Co-IP Troubleshooting

What if I got no protein that interacts with my target protein? Or, what if the signal of interactive protein was too weak?

That may be due to these reasons:

  • The detergent in the lysis buffer might had too high a concentration or too strong.

Decrease the concentration of the detergent, or change it to a mild one. Typically, a strength order of detergents should be:

SDS>Trition>NP40>Digitonin>CHAPS

  • Effect of sub-localization of your target protein in cells.

Change the recipe of the lysis buffer and make the protein released.

  • The protein-protein interactions were too weak or unstable.

Change the antibody to a more affinitive one to capture more of target protein.

Or, apply a expression system to enhance the purity and concentration of target protein.

Or, change origins of samples or optimize the sample treatment to let the sample have more of target protein or interactive proteins.

What if the false positives were too strong or too many non-specific binding proteins were detected?

That may be due to these reasons:

  • The antibody concentration was too high.

Low down the antibody concentration

  • The antibody specificity was not good enough.

Change the antibody to a better one.

If the non-specific binding persists, increase the concentration of NaCl to lower than 1M.

I seem to get low binding of my selecting antibody. What can I do to improve binding?

  • Verify binding/specificity of your antibody to your antigen, e.g., by ELISA.
  • Check the binding of your antibodies to the beads. If the antibodies are not captured and bound to the beads, the immunoprecipitation experiment will not work.
  • Check the amount of beads and sample volume. With reference to the capacity of different beads proposed in the package inserts, increase the amount of beads or the concentration of your antibody during coupling.
  • Increase the incubation time.
  • Try another antibody.