That is quite a size difference between the expected and observed band sizes. A few things come to mind. The first is your protein is getting cleaved or degraded. Do you see any smearing or just one nice band? A second thought is that your antibody recognizes something else in your sample. Even monoclonal antibodies can still have background issues. Do you have a positive control (such as purified protein) that you can run and test?

Hope this helps!

Thanks Amy Klocko i do have a positive control and it’s showing the expected band size. for some cell lines I’m testing I see bands much lower that where I would expect it. Im not seeing any smearing, just bands at much lower positions.

Good to know the positive control works well with the expected band. Any chance of different splice variants leading to protein isoforms?

That's what I was going for, but nothing reported in literature.