You can use up to 0.1% SDS in the TX buffer. Also it's pretty difficult to
"over-transfer". The loading capacities of gel and membrane are in the
same range, so only if you have overloaded the gel will stuff start to leak
through the membrane and out the other side. So, try transferring for
longer. For a biorad mini-gel set up we use 6 hours in the cold-room at
50V constant voltage (about 125mA, depends on temperature), changing the
ice pack every 2hrs. This works fine for proteins up to 260kD. The other
tip is to make your tx buffer just when you put the gel on. This will give
it a chance to cool down while the gel is running, since the mixing of MeOH
and water generates quite a bit of heat. Keeping it all cooler enables you
to run bigger voltages. One final thing is to try nitrocellulose. We've
had problems getting big proteins to stick to PVDF, both 0.22 and 0.45
micron sizes, but they're fine on NC.
Paul
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
Paul S. Brookes. Dept' of Pathology,
University of Alabama at Birmingham,
G004 Volker Hall, 1670 University Blvd.,
Birmingham, AL 35294, USA
205 934 1915 Fax 205 934 1775
brookes at uab.edu2055401028 at mobile.mycingular.net (page,150 ch. max)
http://peir.path.uab.edu/brookes
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