I was running Westerns for many years but mostly from cell lines or primary cells. Recently, we were running Westerns from human tissue which contain traces of blood. We reprobed our 2 membranes with 2 primary antibody (rabbit polyclonal IgG) which recognize 2 proteins of interest (ca. 50 kda each), then we re- probed with anti-rabbit secondary Abs.-HRP conjugated (IgG (H+L) and get ¨¨nice fat¨¨bands at ca. 50 kda. We suspecting that these ¨¨nice fat¨¨ bands are unspecific signal from IgG heavy chain. We need to confirm these by probing the membranes only with secondary antibody but we also get these extra ¨¨nice fat¨bands for protein with ca. 70 kda signal.

My question is, how we can really get rid off these 50kda unspecific bands from our Westerns?

We cannot change the primary antibody which unfortunately recognizes the proteins of interest at 50 kda.

We can change the secondary-HRP conjugated antibody. But which one? Defintely ones that will not recognize heavy chain IgG. For example anti-rabbit F(ab)-secondary Abs-HRP conjugated (which company)? Or maybe TrueBlot sec Abs, that only recognize light chain, not the heavy chain? But I was reading that they are good for IP-WB together, not WB alone?

Or should we get rid off the traces of blood from our tissue before isolating proteins?

I would appreciate any answer, especially from persons that work(ed) with human tissue samples, observed similar problems and can give us good advice/hints.