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: [EMAIL PROTECTED] -----Original Message----- From: Yong Tang [mailto:[EMAIL PROTECTED] Sent: 3/9/2007 (星期五) 12:39 下午 To: [email protected] 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
