Signal stuck on top of seperating gel - (Apr/11/2012 )
I am transfecting pcDNA-constructs into HEK293 cells. After lysation I was usually
able to detect massive signals of my protein (~140kDa) via SDS-PAGE and Western Blotting.
But now it gets frequently stuck on top of the separating gel. Its definitely specific binding, so
i think the transfection efficiency is fine...
I excluded troubles with the sds-loading- and running buffer and also the gels are fine (10%).
Other samples with the same protein are running normal on the gel and don't get stuck.
And I also didn't change anything about the Lysis Buffer (TritonX-100 as detergent)...
I'm really lost right now... :-/
Any suggestions what could cause this?
Can you be a bit more clear by what you mean by "stuck"? Is the sample getting stuck while running (ie: the loading dye is not moving), or are you seeing your protein run higher when blotting? 10% seems a bit high for a protein that large, I would recommend a 7.5% with a 3-5% stacking. Alternatley, have you started using a new batch of SDS-loading buffer that may not be made properly and may not be denaturing as it should be? Have you run samples from these lysates previously? What protocol are you using?