Re: [ccp4bb] precipitation after storage
We routinely use a P200 to pipette drops of protein directly into a small Dewar of liquid nitrogen. The protein forms small BB's with a volume of approximately 30 micro-L each. Pipette slowly, allowing the drops to freeze solid before adding the next one. The frozen BB's can be picked up with forceps or a slotted spoon and stored in a cryovial in the -80 freezer. For future experiments, you can thaw only what you need. I have only seen one protein that couldn't recover from this and could only be crystallized prior to freezing. A systematic study of this procedure is described in Deng et al. http://www.ncbi.nlm.nih.gov/pubmed/14684931 --Andrew On 11/5/10 3:40 PM, Eric Karg harvard...@yahoo.com wrote: Dear all, I'm working on a protein which starts to precipitate after 3-4 days of storage at 4 degrees or room temperature. The storage buffer contains 300 mM NaCl because at lower salt concentrations it also tends to precipitate. Different buffers and adding glycerol did not help although this was not done in a systematic way. Has anyone had similar experiences? Any suggestions to overcome this problem? Thanks in advance! Eric -- Andrew M. Gulick, Ph.D. --- (716) 898-8619 Hauptman-Woodward Institute 700 Ellicott St Buffalo, NY 14203 --- Hauptman-Woodward Institute Dept. of Structural Biology, SUNY at Buffalo http://www.hwi.buffalo.edu/Faculty/Gulick/Gulick.html http://labs.hwi.buffalo.edu/gulick
Re: [ccp4bb] precipitation after storage
A modification of this was done in pcr tubes, and it seemed to work even better: Acta Cryst. (2004). D60, 203-204[ doi:10.1107/S0907444903024491 ] An improved protocol for rapid freezing of protein samples for long-term storage J. Deng, D. R. Davies, G. Wisedchaisri, M. Wu, W. G. J. Hol and C. Mehlin Abstract: Freezing of purified protein drops directly in liquid nitrogen is a convenient technique for the long-term storage of protein samples. Although this enhances reproducibility in follow-up crystallization experiments, some protein samples are not amenable to this technique. It has been discovered that plunging PCR tubes containing protein samples into liquid nitrogen results in more rapid freezing of the samples and can safely preserve some proteins that are damaged by drop-freezing. The PCR-tube method can also be adapted to a PCR-plate freezing method with applications for high-throughput and structural genomics projects. On Mon, Nov 8, 2010 at 8:21 AM, Andrew Gulick gul...@hwi.buffalo.edu wrote: We routinely use a P200 to pipette drops of protein directly into a small Dewar of liquid nitrogen. The protein forms small BB’s with a volume of approximately 30 micro-L each. Pipette slowly, allowing the drops to freeze solid before adding the next one. The frozen BB’s can be picked up with forceps or a slotted spoon and stored in a cryovial in the –80 freezer. For future experiments, you can thaw only what you need. I have only seen one protein that couldn’t recover from this and could only be crystallized prior to freezing. A systematic study of this procedure is described in Deng et al. http://www.ncbi.nlm.nih.gov/pubmed/14684931 --Andrew On 11/5/10 3:40 PM, Eric Karg harvard...@yahoo.com wrote: Dear all, I'm working on a protein which starts to precipitate after 3-4 days of storage at 4 degrees or room temperature. The storage buffer contains 300 mM NaCl because at lower salt concentrations it also tends to precipitate. Different buffers and adding glycerol did not help although this was not done in a systematic way. Has anyone had similar experiences? Any suggestions to overcome this problem? Thanks in advance! Eric -- Andrew M. Gulick, Ph.D. --- (716) 898-8619 Hauptman-Woodward Institute 700 Ellicott St Buffalo, NY 14203 --- Hauptman-Woodward Institute Dept. of Structural Biology, SUNY at Buffalo http://www.hwi.buffalo.edu/Faculty/Gulick/Gulick.html http://labs.hwi.buffalo.edu/gulick
[ccp4bb] precipitation after storage
Dear all, I'm working on a protein which starts to precipitate after 3-4 days of storage at 4 degrees or room temperature. The storage buffer contains 300 mM NaCl because at lower salt concentrations it also tends to precipitate. Different buffers and adding glycerol did not help although this was not done in a systematic way. Has anyone had similar experiences? Any suggestions to overcome this problem? Thanks in advance! Eric
Re: [ccp4bb] precipitation after storage
Hello Eric, Does your protein also precipitate at lower protein concentrations? In isolated cases, we've had protein stocks precipitate overnight at 4 degrees, and the only way around it was to store them diluted, and concentrate right before any experiments/crystallization trials. In two cases, the 'precipitation' in the protein stock appeared to be showers of microcrystals... Filip On Fri, Nov 5, 2010 at 1:40 PM, Eric Karg harvard...@yahoo.com wrote: Dear all, I'm working on a protein which starts to precipitate after 3-4 days of storage at 4 degrees or room temperature. The storage buffer contains 300 mM NaCl because at lower salt concentrations it also tends to precipitate. Different buffers and adding glycerol did not help although this was not done in a systematic way. Has anyone had similar experiences? Any suggestions to overcome this problem? Thanks in advance! Eric -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] precipitation after storage
Hello Eric, I would try not to store a protein for 3-4 days and set up drops as quickly as possible. 3-4 days can be a long time for a sensitive protein. If you expressed too much, add 15% glycerol and store the protein at -80deg. Gel-filtrate the solution and re-concentrate before the next use. Tim On Fri, Nov 05, 2010 at 08:40:51PM +, Eric Karg wrote: Dear all, I'm working on a protein which starts to precipitate after 3-4 days of storage at 4 degrees or room temperature. The storage buffer contains 300 mM NaCl because at lower salt concentrations it also tends to precipitate. Different buffers and adding glycerol did not help although this was not done in a systematic way. Has anyone had similar experiences? Any suggestions to overcome this problem? Thanks in advance! Eric -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A signature.asc Description: Digital signature
