smearing at top of resolving gel - SDS-PAGE (Jan/06/2007 )
I'm having some issues with SDS-PAGE. I am trying to separate proteins of 190 kDa and 50 kDa for specific detection using western blot. The gel (8% resolving) appears to set appropriately, after degassing and layering with water. The gel front appears to migrate well. However, after transfer or coommassie staining of the gel, the higher molecular weight protein is detected as a smear (this also appears at higher molecular weights on the gel), but the lower protein appears as a distinct band on western blot and also lower molecular weight proteins appear well resolved on the gel. Has anyone experienced this previously, and can you give me some advice? Would be greatly appreaciated.
as I understand you like to get a clear band for the 190 kDa; if it is a membrane protein, it is perhaps O- and N-glycosylated which cause the smearing; to circumvent this, deglycosylate (both O +N) with appropriate enzymes (f.i. from NEB) before SDS-PAGE