quantitative gel-shift assays

martin LEACH 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

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.


--
.....          Martin Leach                Email:leach at bu.edu
   _|____      Dept. of Pharmacology       Phone: (617) 638-5323
   / o  /      Boston Univ. School of Med. Fax:   (617) 638-4329
 _/  |-/__==/  80 E. Concord St. (L603)
(BULLDOZER) \_ Boston MA 02118            "Not the old underpants
               USA                                on your
                                             head.....WIBBLE"

My homepage: http://www.tradesmart.com/martin/Leach.html
Webmaster of the Biotech Rumor mill: http://www.tradesmart.com/rumor



More information about the Methods mailing list