protein competitive binding

Dr Engelbert Buxbaum engelbert_buxbaum at hotmail.com
Wed Mar 5 03:59:37 EST 2003


fisher wrote:

> Hi, I am still new in the molecular biology field, and I just got a project
> that needs me to test whether two proteins can bind competitively to the
> third protein in E.coli.
> 
> All the experiments I can think of right now is to purify those proteins,
> and use non-denaturing gel to study the complexes.  Are there any other
> common method that people usually use to study such competitive binding??
> 
> One of my concern is these proteins may not even form complexes in vitro.
> So are there any good in vivo ways to do it??


At least in vitro one possibility would be FRET (fluorescence resonance
energy transfer). The third protein would be labeled with a fluorescenct
dye, one of the other with a second dye, that has an absorbance maximum
identical to the emmission maximum of the first.

If that protein binds, the dyes will get into close proximity and FRET
will occur, that is, exitation with lamda(ex1) will result in emmission
at lamda(em2). 

Addition of another protein that competes for binding will reduce this
effect in a concentration dependent manner. Afffinities for both
proteins can be derived in the usual way.

Dyes with large Stokes-Shifts are easiest to use for this purpose.
Molecular Probes (www.probes.com) supply all the info you will ever need
for fluorescent labeling.



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