running a 6% SDS gel - (Mar/13/2008 )
we are trying to separate two proteins which are about 200-250 kDa by SDS-PAGE (CBP and NCoR). We use 6% separating gel and Dual Color from Biorad as marker. It is a big gel and I run it until the front goes out (a day long). However, after 3-4 hours the marker disappears or the bands shift place. We run at 85V until the end of stacking part and then 120V. We also tried to run it at 4 degree but there was no improvement. The buffers, reagents and the marker are okay since nobody has problems in running 8 or 10% gels (small and big ones).
I would appreciate any suggestion or recipe.
we used 3.5-5% (with 0-8M urea) gradient sds-page to separate myosin heavy chains. the urea helps sharpen the banding.
i don't know if it will make too much of a difference but we use the neville buffer system for our gels. it should work as well with the laemmli system.