Band-shift assay

Karen Spink pdxkgs at pdn1.gene.nottingham.ac.uk
Thu Feb 5 07:52:26 EST 1998


I am very confused!
Recently, I have been trying to purify a DNA-binding protein from yeast and
have constructed a specific DNA affinity column, attaching the binding site
for my protein to CnBr-activated Sepharose. I bound some of my partially
purified protein extract to this column and was successfull in retrieving a
band-shift with one of the fraction. SDS-PAGE informed me that I now had a
dozen or so bands in this fraction, so I re-applied this fraction to the
column and eluted with a narrower range of salt concentrations, but my
protein has now DISAPPEARED! It is neither in the flow through nor any of
the fractions. I have read in a couple of publications that loss of
activity in a band-shift can occur with a highly pure DNA binding protein
and that activity can be regained by the addition of 1mg/ml casein to the
binding reactions. I am hoping someone can enlighten me on this phenomena. 

1) Does this really work ?
2) Why does this work, (BSA supposedly does not have this effect on activity)
3) Is it better to use casein or B-casein
I do not really understand how the addition of casein, rather than BSA, can
affect activity in this way and am hoping not to have to do the whole
purification thing again

Thanks very much for your time,

Karen Spink



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