[ccp4bb] ´ð¸´: [ccp4bb] Removal of bacterial Hsp7 0 contaminant from recombinant protein

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CCP4bb <-- 2007 <-- March 2007 <-- 09 March 2007

Previous message:
Subject: Re: ANIS and SHELXL
From: "Arti S {- dot -} Pandey" artis {- at -} CHEMISTRY {- dot -} MONTANA {- dot -} EDU
Date: 2007-03-09

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Subject: Re: Removal of bacterial Hsp70 contaminant from recombinant protein
From: Brad Bennett saint_genevieve {- at -} ATT {- dot -} NET
Date: 2007-03-09

Subject: ´ð¸´: Removal of bacterial Hsp7 0 contaminant from recombinant protein
From: "Wang, Yeming (NIH/NIEHS) [F]" WangY2 {- at -} NIEHS {- dot -} NIH {- dot -} GOV
Date: 2007-03-09

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: wangy2@niehs.nih.gov

-----Original Message-----
From: Yong Tang [mailto:liutang19@GMAIL.COM]
Sent: 3/9/2007 (ÐÇÆÚÎå) 12:39 ÏÂÎç
To: CCP4BB@JISCMAIL.AC.UK
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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CCP4bb <-- 2007 <-- March 2007 <-- 09 March 2007

Previous message:
Subject: Re: ANIS and SHELXL
From: "Arti S {- dot -} Pandey" artis {- at -} CHEMISTRY {- dot -} MONTANA {- dot -} EDU
Date: 2007-03-09

Next message:
Subject: Re: Removal of bacterial Hsp70 contaminant from recombinant protein
From: Brad Bennett saint_genevieve {- at -} ATT {- dot -} NET
Date: 2007-03-09