I am having the following problem after western blot transfer, and I am not sure what the problem is...
I run the gel at 130 V for 90 mins using a NC membrane and transferred using the iBlot3 machine using the midi transfer stack. The first top membrane looks completely fine, but the second top membrane looks so weird. Both lower membranes look fine.
Any help or advice would be appreciated!
Are these the same blot but stripped and restained? If so, I think it may have to do with the antibody you are using on the bad image. If not, I say try it again.
The transfer is not the issue in the second top blot, in the first blot however there is some transffer-related issues, and that big empty spot suggests that. For the second top blot you could optimize your blocking protocol, its a really high background. Wish u the best with your blots! Sincerlay, Vatamanu.