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

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: rescuing crashing-out protein eluted from Nickel column
From: Valerie Guillet Valerie {- dot -} Guillet {- at -} IPBS {- dot -} FR
Date: 2008-02-15

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Subject: Re: rescuing crashing-out protein eluted from Nickel column
From: "R {- dot -} M {- dot -} Garavito" garavito {- at -} MSU {- dot -} EDU
Date: 2008-02-15

Subject: Re: rescuing crashing-out protein eluted from Nickel column
From: "Mathews, Irimpan" iimathew {- at -} SLAC {- dot -} STANFORD {- dot -} EDU
Date: 2008-02-15

Hi Jacob,

You could try keeping some inhibitor or other small molecules that could bind to the protein in the test tube. This may help stabilizing the protein as soon as it comes out of the column. One of my friends had to use an inhibitor during the whole purification procedure (luckily this compound was very cheap).

best wishes,
Mathews

________________________________

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Wong
Sent: Thursday, February 14, 2008 4:26 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] rescuing crashing-out protein eluted from Nickel column

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.

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: rescuing crashing-out protein eluted from Nickel column
From: Valerie Guillet Valerie {- dot -} Guillet {- at -} IPBS {- dot -} FR
Date: 2008-02-15

Next message:
Subject: Re: rescuing crashing-out protein eluted from Nickel column
From: "R {- dot -} M {- dot -} Garavito" garavito {- at -} MSU {- dot -} EDU
Date: 2008-02-15