protein concetration

[Alex Morla]

In article <3tsnc8$3ou at diplomatic.passport.ca>, scontz at passport.ca (Marc
Visconti) wrote:

> Hello 
>  
> 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 
  Assistant Professor                                                         
  Department of Pathology                                       
  The University of Chicago