A solution may be to change the species of one of the antibodies, either the one used for pull-down or the primary detection antibody.

For example, if you are using a mouse monoclonal antibody to pull down the protein of interest and your primary detection antibody is also a mouse antibody, then your secondary anti-mouse antibody will detect the mouse mAb used for the pull-down.

Another solution would be to use direct detection of the antigen by the primary antibody carrying a fluorescent label or detection enzyme, to avoid the need for a secondary antibody (with some resulting loss of sensitivity).

You can try doing non-reducing condition if you already haven’t. It will help to separate the heavy chain and your target protein. I have tried as my target protein was at ~ 50kDa. I did both reducing and non-reducing and in reducing condition, there was still an overlap.

Later I also overexpressed my target protein and pulled down with the tag which helped me to separate in reducing condition as well because the size increased to ~ 66 kDa.

Good luck :)

Adam B Shapiro Thank you for the suggestion. I will give it a try.

Fabiha Farzana Thank you Fabiha, Do you mind sharing a protocol or a published paper that has that protocol which I can follow and see? Would like to try the native condition since my protein size is same as yours. Also thank you for sharing the idea about overexpressing with a tag. I'll keep that in mind. Thank you