Non-specific Binding in Immunoprecipitation (IP)

Possible Problems & Solutions

A. Non-specific binding of proteins to antibody

If there are too much proteins binding non-specifically, then try decreasing the amount of sample loaded onto the beads. You can also pre-clear the lysate by pre-incubating the prepared lysate with the beads before commencing with the immunoprecipitation. This should clear the lysate of any proteins that are binding non-specifically to the beads. Some researchers also use an irrelevant antibody of the same species of origin and same Ig subclass to pre-clear the lysate.

B. Antibody used contains antibodies that are not specific enough

Use an antigen affinity purified antibody, preferably pre-adsorbed.

C. Non-specific proteins are binding to the beads

Beads are not pre-blocked enough with BSA. Make sure BSA is fresh and incubate fresh beads for 1 hour with 1% BSA in PBST. Wash 3-4 times in PBS before using them.

D. I experience non-specific binding in my immunoprecipitation experiment.

Use more stringent washing buffer for washing.
Add a non-ionic detergent (Tween-20 or Triton X-100) to the washing buffer, in concentrations between 0.01–0.1%.
If the beads are blocked before precipitation, add identical blocker to the washing buffer.
Increase the number of washing.
Prolong the washing time.
Decrease incubation time (beads and sample).
Try the indirect method.
Decrease the immunoprecipitation antibody concentration.
A pre-clearing step is needed to remove molecules that non-specifically bind to the protein A/protein G/protein L or the beads themselves.