Wet Transfer Problem - Front cassette is 'blury' (Feb/23/2005 )
In transferring my western in Tris 3.03g /Glycine 14.4g pH 8.3 onto PVDF at 110V constant for 1 hour on the Biorad mini system the front cassette does not seem to be transferring properly.
The rear casette give good clear banding yet the membrane in the front casette, probed and blocked with the other membrane is blargh. Just looks very swirly.
I know it must be in the transfer as the marker is running striaght on the gel yet not transferring in the front casette. It's happened the last 3 times I've run gels.
I'm doing "black to back - protein's run to red" and the black side of the casette is to the rear of the tank.
No matter how much ice I surround the tank in, it begins to boil at 30 mins. I used to run my transfer in the cold room but now the electrical company has banned us from using a 'heat producing item' in there. I don't know if this is my problem. I chill the buffer overnight and am unsure how to get the reaction 'cooler' if this is the problem.
Several people I know have had probelems transferring two cassettes in one tank. Separate tanks might help.
In terms of your buffer heating up, if you are transferring at 100 V for one hour or 2 the milliamps should be between ~200 and 450 (450 mamp being achieved at the end of the transfer). If the buffer is really heating up during this period there is simply a problem with your buffer. Do you add methanol, this will help keep it cool.