Hi Yong,
Perhaps try one of these:
1. Wash with ATP+Mg when bound on the lMAC column (to get it to
release your protein).
2. Wash with 0.1% Triton when bound on the IMAC column.
3. Hydrophobic interaction chromatography.
4. Wash with 0.5 GuHCl while bound on the IMAC column (pray!!)
On a sad note, the protein will quite often aggregate when it is
released. There is a reason why the only sign of target protein you
see is stuck in a chaperone... but at least you have the chance to
scout around for a good buffer to release it into.
Martin
On Mar 9, 2007, at 6:39 PM, 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
.
B. Martin Hallberg, PhD
Molecular Cell Biology Program
Department of Cell and Molecular Biology
Karolinska Institutet
SE-171 77 Stockholm
Sweden
