quantitative gel-shift assays
leach at bu.edu
Sun Mar 23 18:18:17 EST 1997
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
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,
..... Martin Leach Email:leach at bu.edu
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