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Hsp70 is very hydrophobic, and the problem mentioned by you is quite common in many affinity based purifications. I suggest that you load your sample on an HIC (say, phenyl sepharose) and elute with a gradient from high-salt to no-salt. Hsp70 typically elutes close to no-salt buffers on the HIC chromatography. Shekhar Mande On Fri, 9 Mar 2007, Yong Tang wrote: > 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 > >