Well-retention of protein/DNA complex

jack v052myen at ubvmsb.cc.buffalo.edu
Sat Feb 20 00:28:00 EST 1993


Dear Net:

	I am examining protein/DNA complex formation by means of a mobility 
shift assay in which protein and DNA are pre-incubated, then placed on a 4-6%
acrylamide gel, with 0.5x TBE running buffer.  In general the system works
very well, but I get well-retention of protein/DNA complex if the protein
concentration exceeds a certain level.  I would like to know if anyone has 
hints about getting this material into the gel.  From experience, I know that
the following helps: lower percentage acrylamide, lower crosslinking ratio
of bisacrylamide:acrylamide, thicker gels (more surface area in the well).  In
addition, literature suggests that basic proteins may be hang-up if the buffer
is too acidic; any suggestions on a more alkaline buffer?  To my knowledge, 
this retention is not due to protein aggregation or size of the protein in this
case.  As samples loaded first and last have similar well-retention, I don't 
believe the hang-up has anything to do with "dead-time" in the well prior to
the application of current.
	Any suggestions would be appreciated.  If possible, please reply by
Email.  If a number of suggestions are received, I will edit and repost them to
this group.

	Thanks,

	Jack Welch
-----------------------------------------------------------------------------
John J. Welch				Roswell Park Cancer Institute
v052myen at ubvms.cc.buffalo.edu		666 Elm Street
					Grace Cancer Drug Center, Rm 233
					Buffalo, NY  14263  USA
					(716) 845-3233



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