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 <[EMAIL PROTECTED]>:
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
e-mail: [EMAIL PROTECTED]
homepage: http://www.rug.nl/gbb/research/researchgroups/enzymology/index
