Immunoblot (Western Blot) Question - Trying to nut out a question my demonstrator asked (May/22/2005 )
Hey, got asked a question while doing a western in prac the other day. We had just finished running the SDS-PAGE gel for the western, and the demonstrator asked why we shouldn't fix the gel. Im assuming it inhibits the protein from transfering to the nitrocellulose paper, but i can't see the reason explained anywhere (the net or our textbook). Could anyone give me a quick explanation for why fixing the gel would be a bad idea?
depends on what fixative you use.
Your answer was the first thing in my mind, it won't transfer onto the blot. Another thing that comes to mind is that fixing would mask or deform the epitope your antibody was raised against therefore you may not see the signal at the end.
Good luck with it!