amorla at midway.uchicago.edu
Tue Jul 11 08:36:10 EST 1995
In article <3tsnc8$3ou at diplomatic.passport.ca>, scontz at passport.ca (Marc
> I am currently expressing and purifying a recombinant protein, calculated
> 17 kDa, with the pET-30 expression system from Novagen. The system uses a
> hexahistidine stretch and Ni-affinity chromatography for purification. It
> seems that I have a large yeild, and now concentration is a problem. I am
> currently using Ultrafree-20 low binding cellulose filters from Millipore,
> but I seem to lose a large quantity of my target protein. Does anyone have
> any ideas or tricks that I may try to concentrate my protein prep to
> roughly 1 ml? Has anyone tried using dialysis and polyethylene glycol or
> Sephadex? Any help would be greatly appreciated
> Copyright 1995 Elmer Fudd.
> All wights wesewved.
We typically concentrate our protein preps (made by using the Qiaexpress
system -also 6-His and Ni-column affinity chrom.) by placing the protein
in the elution solution (contains 8M urea, pH is 5.9) in a dialysis bag
and placing it over Aquacide III (flake PEG, from Calbiochem). We usu.
conc. the protein prep approx. 10-fold. Then dialyze extensively against
PBS. Seems to work well for our simple proteins (i.e., no Cys's), but
others may be more difficult.
Hope this helps.
<<<<<>>>>> <<<<<<>>>>>> <<<<<->>>>> <<<<<<>>>>>>
Dr. Alex Morla
Department of Pathology
The University of Chicago
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