quantitative gel-shift assays

P.J. Shaw pjs14 at le.ac.uk
Sat Mar 8 09:24:36 EST 1997


I have two E.coli derived homeodomain peptides (fused to pMALc2) and I 
would like to compare their binding affinities to several types of 
binding sites, all 24bp long. 
To obtain accurate data, what are the critical parameters? 
My proteins are relatively pure (>70%) and I figured that the simplest 
method was to titrate a constant amount of protein against varying 
amounts of labelled probe, assuming that the Phosphorimager data can be 
used to determine directly the concentrations of free and bound probe 
without calculating the specific activities first. Any comments?
 
Philip J. Shaw,
Dept. of Genetics,
Leicester University,
UK.




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