Help with EMSA (using pierce Lightshift kit 20148). - Problems with EMSA using pierce Lightshift kit (Jan/11/2007 )
I am doing EMSA (using pierce Lightshift kit 20148). I use biotin-labeled 30 bp DNA duplex that was incubated with a crude extract of E. coli (BL21 DE3) expressing a target His6-tagged regulatory protein. In those experiments I could see shift bands in my controls that contain just crude extract without protein (containing only the vector =No insert coding for my regulatory protein) and even by using crude extract without DNA!!!. I also got a signal when adding another chemiluminescent substrate to the membrane (AP), so I must have “endogenous-like biotin” in my sample….
Does any one use the Pierce Lightshift EMSA kit with crude extract of E. coli ? and face this problem too ?
If you're seeing a shift band is it likely to be endogenous biotin or just non-specific protein-DNA interactions? What are you using in your binding buffer to stop non specific interactions e.g. poly dIdC or salmon sperm DNA? And what are you blocking and washing your membrane in? Also have you tried to visualise the protein possibly in the membrane using a His tagged ab? It might be worth cleaning up the extract by IP using a anti-His ab and using an irrelevant His-tagged protein as a control in your experiments.