cell lysate PAGE & blotting problem? - (Apr/02/2008 )
I lysed my stable cell line and there is no prior purification, and I run them on 12.5% gel ( stacking is 5% ), but as there are thousands of proteins including my own recombinant one, I see a blue lane (from top to bottom) in commassie blue and a brown lane in silver nitrate staining.
I can not have any better resolution as the number of exsiting proteins in lysate is unduely large so they cover nearly every molecular weight so they seem to be sticking together after staining.
how can I have a good SDS-PAGE for whole cell lysate??
you can improve resolution by reducing the sample load.
you can also increase the length of your gel (need different apparatus).
if you also need to sharpen your bands then you can run a gradient gel.