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Re: [ccp4bb] Off topic: General rule for maximum flow rate for affinity column?

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999
Previous message:
Subject: MAX-lab beamtime Deadline March 14th 2008
From: Thomas Ursby thomas {- dot -} ursby {- at -} MAXLAB {- dot -} LU {- dot -} SE
Date: 2008-02-29
Next message:
Subject: Re: anomalous signal of Mn and Ca ions
From: Stephen Graham stepheng {- at -} STRUBI {- dot -} OX {- dot -} AC {- dot -} UK
Date: 2008-02-29


Subject: Re: Off topic: General rule for maximum flow rate for affinity column?
From: "Mark J {- dot -} van Raaij" vanraaij {- at -} USC {- dot -} ES
Date: 2008-02-29

In general, I would wherever possible bind the protein in batch mode
- 15-30 mins with occasional swirling.
And then pour in a column, wash and elute. If washing and eluting is
very slow, I would apply a little pressure that one time, but the
next time make sure to choose a wider column.
Another common "mistake" is to take too much resin - the minimal
amount to bind the protein should be used, any more will usually lead
to more impurities upon elution.
Mark J. van Raaij
Dpto de BioquĂ­mica, Facultad de Farmacia
Universidad de Santiago
15782 Santiago de Compostela
Spain
http://web.usc.es/~vanraaij/



On 28 Feb 2008, at 23:26, Eric Dollins wrote:

> Dear protein purifiers,
> Off topic question: Is there a general rule for how fast you can load,
> wash and elute from affinity columns, e.g. glutathione agarose? The
> product insert from Sigma says load under gravity flow. For the
> volume of cell lysate I have, gravity loading would take an
> excruciatingly long time. I want to hook up a peristaltic pump to
> speed things along, but don't really have a feel for just how fast one
> can load a column in general (I realize this is also dependent on the
> construct, the buffer, etc). What about the subsequently wash or
> elution?
> Thanks for help
> Eric
>
>
> --
> D. Eric Dollins, Ph.D.
> C266 LSRC, Research Dr.
> Duke University Medical Center
> Durham, NC 27710
> (919) 681-1668, d.e.dollins@gmail.com


CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999
Previous message:
Subject: MAX-lab beamtime Deadline March 14th 2008
From: Thomas Ursby thomas {- dot -} ursby {- at -} MAXLAB {- dot -} LU {- dot -} SE
Date: 2008-02-29
Next message:
Subject: Re: anomalous signal of Mn and Ca ions
From: Stephen Graham stepheng {- at -} STRUBI {- dot -} OX {- dot -} AC {- dot -} UK
Date: 2008-02-29



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