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GELSHIFT HELP, No competition

Eric Hanson Eric.Hansson at medkem.umu.se
Thu Nov 30 05:40:44 EST 1995


I am very,very confused. I am asking if my recombinant transcription 
factor will bind a putative binding site found in an dsDNA 27 mer 
oligo. What I do: end label oligo with 32P, desalt over Biospin 6 
column to remove most of unincorporated hot label, do gelshift with 
(1) crude soluble bacterial extract without expression plasmid 
(Induced with IPTG), to serve as control and (2) crude soluble 
bacterial extract with expression plasmid (Induced with IPTG 
and Wstern confirms it is present and soluble). I see a clean shift 
with extract containing the transcription facor and nothing in the 
control extract. So far great. However, I cannot compete the band 
with 200 fold excess cold probe (added at the same time as hot 
probe). The total probe concentration should be higher than the 
transcription factor on a molar ratio. The band does not compete with 
greater than a 100 fold excess cold ATP ensuring that any 
contaminating hot ATP is not binding. Does anyone have any ideas on 
what is going on? Any help very much appreciated.



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