Western blues

Paul S. Brookes. brookes at uab.edu
Mon Jun 25 12:42:28 EST 2001


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.edu
2055401028 at mobile.mycingular.net (page,150 ch. max)
http://peir.path.uab.edu/brookes
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