John Forsberg, on 30 November 2012 - 08:19 PM, said:
In our system, 100V would probably give an amperage of about 700-800 mA, so it might still be getting quite warm (if I set constant voltage instead of amperage, it's usually ~40V). Are you transferring in a cold room or fridge or at room temp.? I didn't meant to make it sound like everything must be fine if the markers are okay, just that it's encouraging. The only way to tell if any parts of your gel distorted is to probe with your antibodies (or you could do a Ponceau S stain for total protein on your membrane and look for lane/band distortions there, wash off the stain, then probe).
I probed the membrane today, and I got the specific bands i want.There is no distortion of lanes. However, my wild-type band is also shown in all my mut and non-transfected lanes, but not in the empty lane. My mutant band is only shown in my mutant lanes. Can this be caused by the transfer at high-temp (because i don't think i contaminate my samples....)?