Hey Yong... add 10 mM ATP + 2.5 mM MgCl2 in your purification buffer and wash it off during your affinity step...this has worked for me (of course make sure that you're not unlucky and your protein doesn't elute during this step; don't worry it shouldn't) if that doesn't work, maybe try changing some things on the cell growth and expression end, like induction at lower temperature or a different BL21 strain like pLysS or STAR
HTH Brad -- Brad C Bennett Postdoctoral Research Assistant Department of Biochemistry, Cellular and Molecular Biology (BCMB) University of Tennessee-Knoxville 865-974-3045 [EMAIL PROTECTED] -------------- Original message ---------------------- From: Yong Tang <[EMAIL PROTECTED]> > RE: Removal of bacterial chaperone Hsp70 contaminant from recombinant > protein preparation > > Dear all, > > I have a protein expressed at 37C for 3 hours in BL21 DE3 and purified > with sub-stoichiometric amount of apparent Hsp70 contaminant even > after exhaustive affinity (GST-fusion or His-tagged), ion-exchange and > sizing column steps. I would like to know if you have a > well-established protocol for getting rid of such a contaminant. I > asked around and was told to try adding ATP at certain(?) stage of the > purification. > > I would much appreciate your input. > > Many thanks! yong @ the Wistar Institute
