I have begun 2D work on human fluids. I keep running into a problem of some proteins not entering the 2nd dimension.
In this picture i ran 10ug of protein in rehydration buffer (20mM tris, 8M urea, 4%CHAPS 0.5% ampholyte (3-10) and 50mM DTT) (overnight 14hours)
Total focussing was for 25kVhr on a 7cm pH3-10 IPG.
Second dimension equilibrated 1) in DTT and 2x in iodoacetamide) the SDS-PAGE on 8-16% tris-hcl gel.
Possibly they are some high MW proteins? or is there something i might be forgetting?