Protein Degradation after overexpression - (Jun/24/2010 )

Dear colleagues,

I am trying to overexpress (CMV promoter) a transcription factor with FLAG/HA-Tag in HEK293T cells. The problem is that I only get degradet protein: When I load a Western, I see I weak band at the expected 190 kDa (=size of full length protein) but a lot of smear below (down to 40 or 50 kDa). I see that smear especially after overexpression (only weak smear without overexpression) when I use monoclonal antibody for the protein or an antibody against the HA-Tag.

Does anybody have an idea? I think the degradation doesn't come from the lysis conditions. I think the overexpression is the problem. Could another Tag help?

Best and thanks to you all,

Daniel

sorry, i don't understand what you mean by overexpression. how do you define that?