Yong,
I used Tris 10mM, MgCl2 10mM, ATP 5mM (adjust to pH=8.5, w/o buffer, it would be very difficult to adjust the pH value) to wash the resin (10-20 column volume) during on-column wash, and it works.
---------------------
Yeming Wang, Ph.D.
Laboratory of Structural Biology: Macromolecular Structure Group
National Institute of Environmental Health Sciences
National Institute of Health
Mailing Address: Street Address:
NIEHS, MD F3-05 NIEHS, Building 101, Room F363
P.O. BOX 12233 111 T.W. Alexander Drive
RTP, NC 27709 RTP, NC 27709
Tel (o): 919-316-4634
E-mail: [log in to unmask]
-----Original Message-----
From: Yong Tang [mailto:[log in to unmask]]
Sent: 3/9/2007 (ÐÇÆÚÎå) 12:39 ÏÂÎç
To: [log in to unmask]
Subject: [ccp4bb] Removal of bacterial Hsp70 contaminant from recombinant protein
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! ¨Cyong @ the Wistar Institute
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