I am currently trying to isolate a protein that is above 800kDa, a fairly large protein. I have done the SDS PAGE and membrane transfer a few times. After the membrane transfer, each time I stain the membrane (Ponceau & Coomassie) I get no bands. This meant that transfer time is not sufficient (which is one of the problems I am working on).
But the main problem is that when I've stained the gels (Coomassie) I only sometimes see bands on the gel. When I did a 6% gel, I saw a few bands at the top of the gels, but only in a couple of the lanes. When I did a 4% gel, I didn't see any bands on the gel at all, but there was staining at the bottom of the wells (I also stained the stacking gel this time).
Each time I ran the gel at 200V for 1.5 to 2 hours.
Any help of suggestions would be very much appreciated.