UV Crosslinking Protein/DNA

Linda M Ritter lritter at magnus.acs.ohio-state.edu
Mon Mar 6 14:57:28 EST 1995


Help!  We have a novel protein which we would like to UV crosslink to a BrdU 
incorporated probe so as to determine the molecular weight.  The DNA binding 
site is on a 60 bp fragment of DNA.  I have a 15 bp primer and, using Klenow, 
am body labeling with P32 alpha dCTP.  After binding and running on native 
polyacrylamide, I find that there are many extra complexes, so many in fact 
that it is impossible to pick out which is the complex of choice.

I do not know if this is a problem with the BrdU, or with misincorporation of 
nucleotides.  If anyone regularly does this type of procedure, could you please
help me with a little advice.  No one in our lab (or building for that matter) 
has ever done this procedure.  Thank you for your response.

Linda M. Ritter
James Cancer Hospital and Research Institute
The Ohio State University
Columbus, Ohio

e-mail  lritter at magnus.acs.ohio-state.edu



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