Quick navigation:        Home   |    Site Map   ||    References   |    Biography   ||    Copyright   |    Other copyright   |    Contact us   |   
Protein structure
 

Re: [ccp4bb] rescuing crashing-out protein eluted from Nickel column
 

Basic tutorials: 		 
 

CCP4bb navigation

CCP4bb <-- 2008 <-- February 2008 <-- 15 February 2008
Previous message:
Subject: Re: counting constraints?
From: Ian Tickle I {- dot -} Tickle {- at -} ASTEX-THERAPEUTICS {- dot -} COM
Date: 2008-02-15 		Next message:
Subject: Re: rescuing crashing-out protein eluted from Nickel column
From: Artem Evdokimov artem {- at -} XTALS {- dot -} ORG
Date: 2008-02-15


Subject: Re: rescuing crashing-out protein eluted from Nickel column
From: Tommi Kajander tommi {- dot -} kajander {- at -} HELSINKI {- dot -} FI
Date: 2008-02-15
first aid panic rescue might be to quickly add more salt.
(e.g. in couple of steps to 0.5 M-1 M NaCl and see if it clears up).

then add EDTA/or/and dialyse. (have to get teh salt out probably...)

the Ni-ions leaking out are a likely cause. among others.

tommi


Quoting José Trincão :

> Hi Jacob,
> try adding a bit of edta (1mM) to remove any Ni that might come off the
> column. You could also add some DTT or bME to keep cysteines reduced
> (careful, add only after the edta!). In my experience the gradient did
> not work very weel because the protein with have a lot of impurities.
> You can also think about adding a different tag (GST is usually helpful
> in keeping proteins soluble).
> Hope this helps!.
>
> Jose
>
> Jacob Wong wrote:
> > Dear all,
> >
> > I just ran into this problem and would like to see if I could get some
>
> > helpful tips before my protein completely crashes out.
> >
> > I have a protein as 6His fusion and it remained bound to the Ni resin
> > with 40 mM Imidazole wash (added to 1XPBS) but then was eluted off
> > with 200 mM (added to 1XPBS). The protein seemed to be highly
> > concentrated in the elution and began to get cloudy right away, with
> > more and more precipitation produced over a matter of minutes. I felt
> > so helpless, didn't know what to do, and then decided to add 5% of
> > glycerol into one of the fractions but that made it even more cloudy
> > (ohh no...).
> >
> > While the protein is dying in the tube, do you have some quick remedy
> > for me? Thanks very much, -J.J.
>
>


--
Tommi Kajander, Ph.D.
Macromolecular X-ray Crystallography
Research Program in Structural Biology and Biophysics
Institute of Biotechnology
P.O. Box 65 (Street address: Viikinkaari 1, 4th floor)
University of Helsinki
FIN-00014 Helsinki, Finland
Tel. +358-9-191 58903
Fax +358-9-191 59940
ProteinCrystallography.org: Copyright 2006-2007 by Quid United Ltd