Re: [ccp4bb] Ion exchange protein lost

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

Previous message:
Subject: Ion exchange protein lost
From: megha goyal mgbiotec {- at -} GMAIL {- dot -} COM
Date: 2010-05-12

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From: intekhab alam faisaldbg {- at -} GMAIL {- dot -} COM
Date: 2010-05-12

Subject: Re: Ion exchange protein lost
From: Jürgen_Bosch jubosch {- at -} JHSPH {- dot -} EDU
Date: 2010-05-12

Hi Megha,

how about your protein is stuck to the top of your column ?
How does your pressure look like before injection and after injection ?
What makes you believe that you have successfully refolded your protein ?

Just some thoughts,

Jürgen

On May 11, 2010, at 10:53 PM, megha goyal wrote:

> Hi all,
>
>
> Our protein is gcsf. We solubilize the inclusion bodies in 8M urea and 0.1M cysteine and then dilute it 1:20 in refodling buffer 0.1% tween 20 pH 8.2. Then we perform concentration using proflux M12 [just concentration and not diafiltration]. Adjust the pH of concentrate to 4.5 and load it to source 15S resin [strong cation exchange]. The problem is we do not recover our protein on performing IEX. HPLC and absorbance reading on concentrate show the presence of our protein. Buffer for loading is 25 mM na acetate pH 4.5 and elution is same buffer with 0.5 M NaCl. No protein is lost in flow thru and even 2M Nacl washing does not show our protein. . Only when we perform NaOH wash we do see some peak but could not analyse it as it is too alkaline and cant run on SDS PAGE or HPLC.
>
> What could be the reason. Where do we lose our protein. Kindly shed some light on this on where shall I be going wrong.
>
> thanks and regards,
>
> meg

-
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab: +1-410-614-4894
Fax: +1-410-955-3655
http://web.mac.com/bosch_lab/

CCP4bb navigation

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

Previous message:
Subject: Ion exchange protein lost
From: megha goyal mgbiotec {- at -} GMAIL {- dot -} COM
Date: 2010-05-12

Next message:
Subject: ligand modelling
From: intekhab alam faisaldbg {- at -} GMAIL {- dot -} COM
Date: 2010-05-12