Re: [ccp4bb] Reg. Protein purification

- Protein crystallography

Main steps:

- Protein purification
- Crystallisation

Special:

- Programs for crystallography
- X-ray detectors

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   - Amino Acids

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

Previous message:
Subject: Re: Reg. Protein purification
From: Roger Rowlett rrowlett {- at -} COLGATE {- dot -} EDU
Date: 2010-01-08

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From: Vin Purp buckybadger97 {- at -} NETSCAPE {- dot -} NET
Date: 2010-01-08

Subject: Re: Reg. Protein purification
From: Ravindra Makde ravimakde {- at -} YAHOO {- dot -} COM
Date: 2010-01-08

you may try partial denaturation during purification steps ( i.e. to include 3 M urea in your gel filtration or ion exchange or N-nta buffers), that may loosen up the interactions without complete denaturation. You may afterward dialyse to remove urea.

r

Ravindra D. Makde

Currently,
Postdoctoral fellow,
Tan Lab,
Dept of Biochem & Mol biology,
The Pennsylvania State University,
University Park, PA, US.
Tel: 814-777-1776 (mobile)
email: rdm19@psu.edu

Scientific Officer E,
High Pressure Physics Division,
Bhabha Atomic Research Centre,
Trombay, Mumbai-400085
INDIA

Tel: +91 22 25593761 (Lab)
email: ravimakde@yahoo.com
ravimakde@gmail.com

--- On Fri, 1/8/10, Sivaraman Padavattan wrote:

> From: Sivaraman Padavattan
> Subject: [ccp4bb] Reg. Protein purification
> To: CCP4BB@JISCMAIL.AC.UK
> Date: Friday, January 8, 2010, 7:41 AM
> Dear all,
> I am trying to express the human protein using bacterial
> expression strain (Rosetta) and purified using Ni-NTA
> affinity purification. The Molecular weight of out protein
> is 47 kDa. In SDS-PAGE, we have seen that 27 kDa contaminant
> protein co-eluted with our protein even at high
> concentration of Imidazole. In Superose 12 column, these two
> proteins eluted as a single peak and its corresponding
> molecular weight suggestive of partial interaction. By mass
> mapping we have found 27 kDa band is an adenylate kinase. Is
> there any specific way to separate adenylate kinase from
> our protein?
>
> Thanks in advance,
>
> Sivaraman Padavattan
>
>
>
>
>

CCP4bb navigation

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

Previous message:
Subject: Re: Reg. Protein purification
From: Roger Rowlett rrowlett {- at -} COLGATE {- dot -} EDU
Date: 2010-01-08

Next message:
Subject: Blob help
From: Vin Purp buckybadger97 {- at -} NETSCAPE {- dot -} NET
Date: 2010-01-08