gel retardation assay

lyang at lyang at
Mon Dec 1 22:36:25 EST 1997

Hi all,
I use the protein including the histag to bind the 5'-end labeled DNA to 
do the gel retardation assay,
but each time, the lanes which has higher concentration of protein are
smeared but the lower concentration not. I make the reaction condition the
same except the concentration of protein. My binding buffer is : 20mM
Tris-HAc(PH7.5), 0.1mMEDTA,0.1mM DTT,50mM NaCl, 12.5%glycerol, 25mM MgAC.
Does anybody know why it is smeared ,and if the histag will affect the
protein-DNA binding? or the reaction condition need slightly modify?
Any idea will be much appreciated.


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