Hello everyone,

Currently, I'm trying to detect five different proteins on a single western blot gel. To do this, I load 5 of the lanes with my sample lysate and I cut it vertically along each of the lanes so that each strip will contain my sample. I do this to avoid cutting into proteins with similar molecular weights. However, as a loading control, I use beta-actin, which has the highest molecular weight out of all the proteins so I cut the blot horizontally. The problem that I've been encountering is with one of the proteins that has a MW close to that of beta-actin, one with a MW of 49kDa and beta-actin with 42 kDa. Using my protein ladder, I have been successfully able to cut it so that I can detect both proteins but it may be hard to do consistently.

I was wondering if there were people who have faced a similar problem and what their solution to it was?

Thank you!