Matt,
I have found the optimum solubility screen to be helpful:
<http://www.ncbi.nlm.nih.gov/pubmed/15333951> Optimum solubility (OS)
screening: an efficient method to optimize buffer conditions for homogeneity
and crystallization of proteins.
Jancarik J, Pufan R, Hong C, Kim SH, Kim R.
Acta Crystallogr D Biol Crystallogr. 2004 Sep;60(Pt 9):1670-3. Epub 2004 Aug
26.PMID: 15333951
Eric
_____________________________________________
Eric A. Toth, Ph.D.
Assistant Professor
Department of Biochemistry and Molecular Biology
Marlene and Stewart Greenebaum Cancer Center
University of Maryland School of Medicine
108 North Greene St.
Baltimore, MD 21201
Email: <mailto:etoth...@umaryland.edu> etoth...@umaryland.edu
Phone: x-410-706-5345
Fax: x-410-706-8297
<http://medschool.umaryland.edu/FACULTYRESEARCHPROFILE/viewprofile.aspx?id=8
032>
http://medschool.umaryland.edu/FACULTYRESEARCHPROFILE/viewprofile.aspx?id=80
32
<http://crystal.umaryland.edu> http://crystal.umaryland.edu
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Matthew Bratkowski
Sent: Thursday, August 12, 2010 2:57 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Suggestions for Reducing Protein Precipitation
Hi.
I am working with a protein that has difficulties staying in solution when
concentrated. The buffers that I have been using contain 20 mM Tris pH 8.0,
5 mM BME (or 2 mM DTT), 10% Glycerol, and NaCl from 50 mM up to 2 M. The
protein seems fine to dialyze into low salt buffer (50 mM NaCl) when dilute,
but seems to precipitate onto an anion exchange column during loading, as
noticed by brown spots on the column and a reduction in yield after the run.
Despite these issues, I have been able to concentrate the protein to about
18 mg/mL after removing glycerol and using 200 mM NaCl and 2 mM DTT.
However, after sitting at 4 C for a few hours, I again notice precipitate in
the concentrated stock. I figure that I will probably just work with more
diluted protein next time, but was concerned that my protein was not stable
enough in this buffer to use for crystallization.
I am seeking advice on finding a better buffer for the protein. Can anyone
suggest whether making subtle buffer changes, such as using HEPES instead of
Tris or KCl instead of NaCl would be advantageous? Additionally, I was
interested I find out what concentration of glycerol or NaCl could still be
added for the protein to still be usable for crystallization, as well as any
additives that will prevent precipitation but not interfere with
crystallization. Also, could someone suggest a good way to test protein
stability in various buffers on a small scale before deciding on conditions
for a large batch.
Thanks,
Matt
