I'm currently trying to do some EMSA using P32. The first two gels looked perfect, with sharp bands and no background. However, all the following ones looked like the blot below. I'm not doing anything different. All my buffers and gels were freshly prepared, and I even produced fresh nuclear extracts to make sure my other batch was not degraded, and it still looks like that. So far, I've repeated it five times, but nothing's changed. Has anyone had the same problem or an idea of what I can do?
How are you exposing/scanning your gel? Do you dry your gel and then expose it? If you're gel is not dry enough when you expose it may be shrinking and warping leading to a messy signal. If it is an instrument issue, maybe the focus of the instrument might need to be corrected. You could increase the pixel count, it may longer to scan but might yield clearer bands.
Ryan Yellamaty Thank you for your answer.
After I run my gel I put it in a 10% ethanol and 10% acetic acid solution for 45 min after which I air dry the gel for 2 h using Whatman (under the chemical hood). For exposure I use a phosphor storage screen and I use a Phosphorimager (Typhoon) to scann my gel after 16h. The gel is not as dry as it would be after using a vacuum dryer but is feels dry to the touch. Due to the solution it will not shrink and so far it also did not warp.
I'm not sure if I can change something at the instrument but I can ask.
- Badges
- Science topic