quantitative gel-shift assays

[martin LEACH]

If you look in Current Protocols (red book) there is a support
protocol in Section 12.4.6 (supplement 15-17 has it at least)
that allows you to obtain equlibria binding constants...

they use footprinting instead of gel-shifts though....

just my thrupenny bit

Martin

P.J. Shaw (pjs14 at le.ac.uk) wrote:
: 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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