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Hi,

What size is the protein and what detergent are you using? Depending on the size of the protein and the size of the detergent micelles you might very well concentrate the detergent during the protein concentration. DDM for example is always concentrated if you have a MWCO less than 100kDa. High detergent concentrations are usually not very good for the protein in the long run.

Cheers,
Ronnie

On Jan 24, 2008, at 9:12 AM, M T wrote:



2008/1/23, Zheng, Lei <[log in to unmask]>:

Hi ccp4ers,

 

Sorry for this out-topic question:

Recently we have a membrane protein expressed, after solubilized with detergent and purified from IMAC, the protein looks beautiful in SEC. However, it completely precipitates after the 2-3 days storage in 4 degree. We supplement 2 mM DTT in the new elute from IMAC, the protein looks happy during weeks at 4 degree. However, it starts to form an invisible aggregate (verified from SEC) during the protein concentration by Centricon. I know this is not uncommon problem for both soluble and membrane proteins and wonder if anyone has any tip and experience to overcome this problem.

The protein pI is 8.6, buffer used is pH 7.6. Glycerol is always present during the purification. We do have high salt (500mM) in the buffer.

 

Thank you for you input in advance,

Lei

 


Ronnie Berntsson

----------
Ph.D. Student
Department of Biochemistry
Groningen Biomolecular Sciences and Biotechnology Institute
& Zernike Institute for Advanced Materials
University of Groningen
Nijenborgh 4, 9747 AG
Groningen, The Netherlands

telephone: +31 50 363 4195
telefax: +31 50 363 4165