u/Cautious_Increase382

Hi everyone, I've been doing Co-IPs to validate hits from my mass-spec data of proximity based labeling. I was using a positive control to validate that the Co-IP worked. The cell lines used for my Co-IP was my parental cell lines which do not have the fusion protein clone, and then I had my stable cell lines which express the fusion protein clone. Both cell lines (parental and clone-expressing cell lines) were co-immunoprecipated with MYC beads and an isotype beads (to test for non-specific binding). The positive control protein showed clear enrichment in the clone-expressing cell line, and no signal in the isotype control beads. However, there is some background in the MYC beads but not in the isotype control beads for the parental cell lines. My initial hypothesis was that it could be because of the positive control protein being a DNA binding protein having non-specific binding and stickiness but my PI said that it would've then showed up in the isotype control, which makes sense. Any advice on why this could happen? Could this be because of improper washing or cell lysis? Any feedback would be appreciated!!