I performed co-immunoprecipitation (co-IP) with mouse anti-GFP antibody and mouse anti-myc antibody. Non-specific bands appeared in all samples in the co-IP experiment. Non-specific bands were observed in all sample groups, including co-IP samples, input samples, and control samples (untransfected samples). Blocking with skim milk and BSA did not solve the problem. Why is this?
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In IP experiments, in order to avoid interference from the light and heavy chains of antibodies, the following strategies can be adopted:
1. Select IP antibodies and IB primary antibodies from different species to eliminate interference caused by cross-reactions;
2. Use anti-light chain secondary antibodies for the IB link to avoid signal interference from heavy chains;
3. If the light and heavy chain signals do not overlap with the target bands, there is no need to optimize the experimental conditions. By adding isotype controls, it can be verified that the source of the additional bands is the light and heavy chains of the antibody.
The answer to this question comes from MedChemExpress (MCE) Technical Support.
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