Hello Matt, did you use a nickel (or similar) affinity column in the purification + is there any beta-mercaptoethanol or DTT in your sample? Nickel can easily 'bleed' off the column and will turn brown when complexed with bME or DTT - this complex can get quite large and even insoluble if enough is present, and would therefore concentrate in your sample.
Cheers Filip On Sat, Sep 6, 2008 at 4:49 AM, Joe Cockburn <[EMAIL PROTECTED]> wrote: > Hi Matt, > I sometimes see a similar thing with my proteins, which definitely don't > possess metal co-factors or prosthetic groups. I found that gel filtration > got rid of it - the browny-yellow stuff came out in the void fraction so I > figured it was aggregated protein. I think it was aggregation via the > his-tags around traces of copper in my sample, which could explain the > brown-ish colour. > What happens if you concentrate the protein in the presence of EDTA? > Joe > > > Hello. > > > > I am working with a protein that turns a yellowish-brown color when it is > > concentrated to around 2 mg/ml or higher in a small volume (a few hundred > > uL). I was wondering if the protein bound a metal or other prosthetic > > group that would give it this color? The protein's color somewhat > > resembles iron binding proteins, but there is no peak in the 400 nm range > > that would suggest heme, and an iron sulfur cluster is not that likely > > since there are only five cysteines in the protein. Proteins with > > structures homologous to the one I am studying bind magnesium, but are > not > > know to bind other metals. Any information about what this color might > > suggest about the protein or how I could analyze possible bound metals or > > prosthetic groups using only a small amount of protein would be helpful. > > > > Matt > > > -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: [EMAIL PROTECTED] http://crg.ubc.ca/VanPetegem/
