Re: [ccp4bb] Concentrating protein

- Protein crystallography

Main steps:

- Protein purification
- Crystallisation

Special:

- Programs for crystallography
- X-ray detectors

Basic tutorials:

   - Chemistry
   - Protein
   - Peptide
   - Amino Acids

Xtal community:

- CCP4BB

CCP4bb navigation

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

Previous message:
Subject: Re: Reminder - still no 3D stereo under OS X 10.5
From: Ben Eisenbraun bene {- at -} CRYSTAL {- dot -} HARVARD {- dot -} EDU
Date: 2008-06-27

Next message:
Subject: Reducer and crystallization
From: Sam samy121 {- at -} HOTMAIL {- dot -} COM
Date: 2008-06-27

Subject: Re: Concentrating protein
From: Roger Rowlett rrowlett {- at -} MAIL {- dot -} COLGATE {- dot -} EDU
Date: 2008-06-27

Guenter Fritz wrote:
> A mild and quick method is to use dry Sephadex G-25. The material will
> swell and take up all the liquid except molecules larger than ca. 5 kDa.
>
>
>> Dear All,
>>
>> we have GCSF protein produced in inclusion bodies. we solubilise it refold
>> it and then concentrate it using proflux system. still the concentration
>> of the protein we get is less and volume is more for us to load in Ion
>> exchange chromatography. is there any simple technique that can be
>> performed in lab without using any hi-fi instrument to concentrate the
>> protein in small volume of buffer. the protein we obtain is about 0.7
>> mg/ml and we get 450 ml solution. our column is 110ml lab scale and we
>> have to work in that only. i have heard of NH4SO4 precipitation. but it
>> requires protein conc more than 1 mg/ml.
>>
>> kindly help me to progress in my experiment.
>>
>>
One of the beauties of ion-exchange chromatography is that it is an
excellent concentration step as well as a purification methodology. It
may take less time and involve less protein loss to pass all the
solution through the IEX column and bind the protein, assuming you have
the protein in a low ionic strength buffer at the appropriate pH.
Elution in a smaller volume can be accomplished by increasing the NaCl
concentration to an appropriate level. In the bad old days before
bacterial overexpression, we used to to this routinely to concentrate a
liter or more of protein extract to 50-100 mL after elution from a
small, high-capacity IEX column.

Cheers,

--
------------------------------------------------------------------------
Roger S. Rowlett
Professor
Colgate University Presidential Scholar
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@mail.colgate.edu

CCP4bb navigation

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

Previous message:
Subject: Re: Reminder - still no 3D stereo under OS X 10.5
From: Ben Eisenbraun bene {- at -} CRYSTAL {- dot -} HARVARD {- dot -} EDU
Date: 2008-06-27

Next message:
Subject: Reducer and crystallization
From: Sam samy121 {- at -} HOTMAIL {- dot -} COM
Date: 2008-06-27