Dear Mohammad, If your protein is purified from insoluble material there could be some DNA in there though if it were stoichiometric your 26 would be >> than your 280, as the former has a much higher extinction co-efficient. A ratio of 2 is could be RNA contamination. I'd also check the mass spec of your protein to see if it has any unusual modification - urea is notorious for cause carbamylation of N-terminal amino groups, and of lysine and arginine residues. What that does to UV I'm not sire sure but irrespective of the UV anomaly, I'd always get the beast mass-specced!
On 7 June 2017 at 14:36, Mohammad Khan <mohdkhan0...@gmail.com> wrote: > Dear all, > > I am working with an exonuclease by refolding it from inclusion bodies > (IBs). I tried various constructs and hosts, but couldn't get it in soluble > form. > > I lyse my cells using a cell disruptor and after solubilizing IBs with > urea, I refold the protein by rapid dilution and get an aggregate and > monomer peak of the same on GFC. and have checked CD as well as activity, > both of which are good. > > My issues is as follows: > > I get a high 260 nm peak while purifying it on GFC. the 260/280 ratio can > reach upto 2. I have tried all means to get rid of watever this > contamination is: cleaned the IBs with 1% Triton X-100, 2 M NaCl, added > Dnase prior to lysis. I have also used methods to remove the DNA from > protein, if that is the contaminating agent. > I am trying to crystallize the protein with no success so far. > Moreover, my thermofluor assays give very low fluorescence. I use Sypro > Orange as a fluorophore. > > Suprisingly, a point mutation in the active site (His to Arg) gets rid of > the issue of contamination and gives me good thermofluor curves. I purify > the mutant also form IBs. > > Can someone suggest what this "contamination" may be? > > Thank you for your time. > > > -- Best wishes Prof. Jon R Sayers, FRSB Tel: +44 (0) 114 2159552 Email: j.r.say...@shef.ac.uk http://www.sheffield.ac.uk/iicd/profiles/sayers
