transfer problem - blue nitrocellulose paper (Nov/24/2008 )
I just did a transfer from an SDS-PAGE gel to nutrocellulse membrane, leaving the loading buffer line on the gel. The entire membrane is blue now, and I wanted to know if anyone else has had this problem, and what causes it. Will it interfere with my immnunoblotting? And I did not stain the gel before transfer.
is there any part of the membrane that didn't turn blue?
did it turn blue during transfer?
is your transfer buffer fresh?
it may be that your bromphenol blue diffused through the gel and only had a small portion in the dye front. this could happen if you have too much bpb in the loading buffer.
may cut the blue line from the gel before transfering.
mdfenko is the master of western blot around here but my experience also might help. I usually stop the gel exactly when the bormophenol blue is just about to leave the gel. now some people let the dye comepletely leave the gel and into the tank buffer but I don't do that because when I run my electroblotter I can make sure my transfer has worked well by checking the last filter. I mean usually bromophenol blue goes to the last filter during transfer and stays there. this gives the filter paper a yellowish green color. possibly you still had some bromophenol blue on left on your gel. now some people lay the gel on the membrane manually but some people like me first release the gel into the transfer buffer and then soak the membrane in the transfer buffer and put it exactly beneath the floating gel and lift them up together. I used to lay the gel manually but I always had to re-lay my gel on the membrane many times because it just didn't spread well on my membrane. this way I usually ended up staining my membrane in different parts.