Re: [ccp4bb] To solve the problem of an extremely asymmetric peak shape obtained from gel filtration chromatography

[Ross Robinson]

It would be sensible to analyse the state of your purified protein further.

  *   (SEC in reasonable salt conc containing buffer – this is standard 
practice)
  *   Analytical ion exchange of purified protein - are there different states?
  *   SEC  (in reasonable buffer) in line with MALS – is there a monomer-dimer 
equilibrium?
  *   Analytical SEC (without further concentration) of the potential dimer 
side of the peak, repeat for monomer side – could give info on potential 
monomer-dimer equilibrium

Cheers,
Ross



From: CCP4 bulletin board  On Behalf Of Javier Gonzalez
Sent: 09 December 2020 14:52
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] To solve the problem of an extremely asymmetric peak 
shape obtained from gel filtration chromatography

[EXTERNAL SENDER]

Hello, I agree with Roger, you should definitely try to increase the salt 
concentration to get rid of non specific binding impurities. And if that 
doesn't work, you can try purifying your protein under denaturing conditions by 
adding one refolding step in the column.
Good luck,
Javier

On Wed, Dec 9, 2020 at 11:26 AM Roger Rowlett 
mailto:rrowl...@colgate.edu>> wrote:
Salt concentrations less than 100 mM can lead to nonspecific adsorption to the 
gel exclusion media, potentially leading to band broadening, and delayed 
elution.  Overloading gel exclusion columns (more than 2-4% Vt) can also lead 
to elution band artifacts. Check these issues first.

Roger Rowlett
Gordon & Dorothy Kline Professor, Emeritus
Department of Chemistry
Colgate University

On Wed, Dec 9, 2020, 9:17 AM  
mailto:sz20203020...@cau.edu.cn>> wrote:
Dear All
There is a 43kd protein purified via Ni-chelating affinity 
chromatography, anion exchange chromatography and gel filtration chromatography 
in sequence. However the chromatogram obtained showed an extremely asymmetric 
peak shape. The aggregation forms of proteins are mainly in the range of 
monomers and dimers(Hepes and low concentration of salt were used as buffers 
for gel filtration chromatography). 5% glycerinum and 1mM Benzamidine 
hydrochloride had been added in order to maintain the stability of the protein 
and prevent it from degrading. But well, all the efforts seem to be useless. We 
wonder if there are any effective measures can be taken to radically solve this 
problem. We would be much indebted for the suggestions you offer.


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--
Dr. Javier M. González
Instituto de Bionanotecnología del NOA (INBIONATEC-CONICET)
Universidad Nacional de Santiago del Estero (UNSE)
RN9, Km 1125. Villa El Zanjón. (G4206XCP)
Santiago del Estero. Argentina
Tel: +54-(0385)-4238352



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