Re: [ccp4bb] Reg. Protein purification

- Protein crystallography

Main steps:

- Protein purification
- Crystallisation

Special:

- Programs for crystallography
- X-ray detectors

Basic tutorials:

   - Chemistry
   - Protein
   - Peptide
   - Amino Acids

Xtal community:

- CCP4BB

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

Previous message:
Subject: Re: Cavity filling
From: Simon Kolstoe s {- dot -} kolstoe {- at -} UCL {- dot -} AC {- dot -} UK
Date: 2010-01-08

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

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

http-equiv="Content-Type">

An anion exchange purification (e.g. Q-Sepharose)
on a small (e.g. 1x5 mL) column with a 10 CV gradient of NaCl is a
pretty good polishing step, and quick. Dilute your protein
sample to <20 mM salt and apply to an anion exchange column at pH
8.0 or so, and elute with a 10 CV gradient of 0-0.5 M NaCl or so. That
nearly always cleans up partially purified protein preps to
crystallography-grade homogeneity. If for some reason you suspect your
protein is basic (pI>8.0), try a cation exchanger (e.g.
SP-Sepharose) at pH 6.0 or so along the same lines.

Cheers.

On 1/8/2010 7:41 AM, Sivaraman Padavattan wrote:
cite="mid:5e4e34921001080441w71287bd2na28f91c7190edfc5@mail.gmail.com"
type="cite">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

Roger S. Rowlett

Professor

Department of Chemistry

Colgate University

13 Oak Drive

Hamilton, NY 13346

tel: (315)-228-7245

ofc: (315)-228-7395

fax: (315)-228-7935

email: rrowlett@colgate.edu

CCP4bb navigation

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

Previous message:
Subject: Re: Cavity filling
From: Simon Kolstoe s {- dot -} kolstoe {- at -} UCL {- dot -} AC {- dot -} UK
Date: 2010-01-08

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