The western blot running phase seemed okay - my lab ran out of running buffer, so the supply company said we could use the transfer buffer instead with an additional amount of sds - we ran a practice gel with this, which worked very well.
Specifically, in this problem run I used nitrocellulose membrane, and one gel was loaded with 72 ug, while the other was loaded with 3 ug of protein in each well.
I stained both membranes with coomassie blue, and while one seemed to retain striking amounts of protein, the other seemed completely empty (so I really did not know what to make of it).
Can anyone tell me why my molecular marker dyes would disappear during a 20 hour transfer at such a low voltage? I've transfered overnight before with no similar problems.
Could the proteins have blown through?
Could the anode and cathode have been switched when the apparatus was loaded?
Could the apparatus have gotten too hot overnight?
Could the molecular markers have just traveled faster (and through) the membrane?
Could there have been some problem with the buffer or gels (both were 10% acrylamide).
Am I likely to have any protein left on the membrane (I'm probing for a low molecular weight protein on one membrane and a high molecular weight on the other).
Thanks for your help!
Edited by fotolilith, 29 July 2004 - 08:06 PM.