first aid panic rescue might be to quickly add more salt. (e.g. in couple of steps to 0.5 M-1 M NaCl and see if it clears up).
then add EDTA/or/and dialyse. (have to get teh salt out probably...) the Ni-ions leaking out are a likely cause. among others. tommi Quoting José Trincão <[EMAIL PROTECTED]>: > Hi Jacob, > try adding a bit of edta (1mM) to remove any Ni that might come off the > column. You could also add some DTT or bME to keep cysteines reduced > (careful, add only after the edta!). In my experience the gradient did > not work very weel because the protein with have a lot of impurities. > You can also think about adding a different tag (GST is usually helpful > in keeping proteins soluble). > Hope this helps!. > > Jose > > Jacob Wong wrote: > > Dear all, > > > > I just ran into this problem and would like to see if I could get some > > > helpful tips before my protein completely crashes out. > > > > I have a protein as 6His fusion and it remained bound to the Ni resin > > with 40 mM Imidazole wash (added to 1XPBS) but then was eluted off > > with 200 mM (added to 1XPBS). The protein seemed to be highly > > concentrated in the elution and began to get cloudy right away, with > > more and more precipitation produced over a matter of minutes. I felt > > so helpless, didn't know what to do, and then decided to add 5% of > > glycerol into one of the fractions but that made it even more cloudy > > (ohh no...). > > > > While the protein is dying in the tube, do you have some quick remedy > > for me? Thanks very much, -J.J. > > -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
