elution of an his-tag protein
sai at topaz.microbio.uab.edu
Tue Jul 23 12:30:46 EST 1996
On Mon, 22 Jul 1996, Falson wrote:
> Keld Sorensen wrote:
> > Try elution with 100 mM EDTA
> > Keld.
> thank you keld, but I forgot to say that our protein is denaturated
> by EDTA or EGTA once solubilized...hum
no...i dont think you want to elute with edta unless your goal is to also
strip your column of the metal it has chelated to it (Ni+ in most
cases...). try 1M imidazole, 0.5 M NaCl (to keep your protein from
aggregating...) and 20 mM tris-HCl ph 7.5 to 7.9. this should elute out
your protein...try atleast 4-5 elutions and check the OD at A280 to
confirm whn your protein has stopped eluting...if you want to optimze the
purity as opposed to optimizing yield, then try a gradient of imidazole
conc...starting at, say, 0.5 M imidazole and eluting with increasing
concentrations...your most cleanest protein should elute out at the higher
imidazole concentrations...after this, if you wanna clean up your column
and regenerate it, THEN do the edta treatment (50mM to 100 mM edta).
remember to re-charge yuour column with your charging solution (100 mM
NiSO4)...hope this helps..cheers...
Arioch (sai at topaz.microbio.uab.edu)
Graduate Student ( and Lord of Chaos...)
Dept. of Biochemistry and Molecular Genetics...
Milieu of Entropy (a.k.a University of Alabama at Birmingham...)
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