UREA in SDS-PAGE - How to make the gel (Aug/08/2006 )
I was trying to prepare standard (Laemmli) SDS-PAGE containing 8M Urea (I did not change the SDS-PAGE recipie, just added urea). Separation gel was fine (however polymerized very fast) but when pouring the stacking gel urea precipitated. I did several attempts but this happened each time.
I guess I did not do this the right way - can anyone tell me how to prepare this gel?
I aimed for 8M urea in gel, buffer and sample.
Maybe I do not need it all these places?
Or maybe urea concentration is too high?
I want to do this because in WB of my transiently transfected cell lysates I see doublets and triplets of my protein of interest and I think this may be due to inproper denaturation of protein.
Any better suggestions of what to do ?
i used to make 7M and 8M urea gels and it worked fine. I didnt add SDS though. Just in place of SDS add Urea and keep everything else the same. I dont think running urea together with SDS makes sense. Maybe if you are worried try to denature in 8M urea first, then SDS sample buffer and then run on usual SDS PAGE. Hope that helps!