Re: [ccp4bb] Tris buffer in cryo protectant
Ursula, After extensive testing, I have found out that in most cases pH changes during flash freezing does not pose a problem. In some cases it can be beneficial. I encourage you to try +/- 2 pH units from your crystallization pH. Sometimes a sub-optimal pH is chosen just because the crystals were obtained the first time at that pH, sometimes it is for strategic reasons, for example to prevent excessive nucleation. The best pH to obtain BIG crystals is not always the best pH to get the best diffraction. Test various pH if you have enough crystals to do that. Enrico. On Fri, 12 Jun 2015 22:47:10 +0200, Ursula Schulze-Gahmen uschulze-gah...@lbl.gov wrote: Does anyone have experience with Tris buffer in cryo protectants? I would expect the pH of the cryosolution to increase a lot during flash freezing which could perhaps destroy the diffraction. I rarely use Tris for crystallization but the current protein really prefers Tris. I would appreciate any comments. Ursula -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
[ccp4bb] AW: [ccp4bb] Tris buffer in cryo protectant
Dear Ursula, just a stupid question: did you try freezing a crystal? There are quite a few crystal structures in the PDB with (a) bound Tris molecule(s), so quite some crystals were not destroyed by a pH shock during freezing. If you tried freezing and saw no/bad diffraction, you should try to take some shots at room temperature to find out whether the diffraction is destroyed by the freezing, or whether your crystal diffracts just badly from the very beginning. There are sleeves you can put over the loop with your crystal to prevent the crystal from drying out. Good luck, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Ursula Schulze-Gahmen Gesendet: Freitag, 12. Juni 2015 22:47 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] Tris buffer in cryo protectant Does anyone have experience with Tris buffer in cryo protectants? I would expect the pH of the cryosolution to increase a lot during flash freezing which could perhaps destroy the diffraction. I rarely use Tris for crystallization but the current protein really prefers Tris. I would appreciate any comments. Ursula -- Ursula Schulze-Gahmen, Ph.D. Project Scientist UC Berkeley, QB3 360 Stanley Hall #3220 Berkeley, CA 94720-3220 (510) 643 9491
[ccp4bb] Tris buffer in cryo protectant
Does anyone have experience with Tris buffer in cryo protectants? I would expect the pH of the cryosolution to increase a lot during flash freezing which could perhaps destroy the diffraction. I rarely use Tris for crystallization but the current protein really prefers Tris. I would appreciate any comments. Ursula -- Ursula Schulze-Gahmen, Ph.D. Project Scientist UC Berkeley, QB3 360 Stanley Hall #3220 Berkeley, CA 94720-3220 (510) 643 9491
Re: [ccp4bb] Tris buffer in cryo protectant
Might be worth trying to see if your protein will still crystallize in a mixture of tris and TAPS buffer? The pKa of the latter is very close to tris, but goes in the opposite direction with temperature - a roughly 3:2 TAPS:tris mix should have minimal pH change on freezing. Tristan Croll Lecturer Faculty of Health School of Biomedical Sciences Institute of Health and Biomedical Engineering Queensland University of Technology 60 Musk Ave Kelvin Grove QLD 4059 Australia +61 7 3138 6443 This email and its attachments (if any) contain confidential information intended for use by the addressee and may be privileged. We do not waive any confidentiality, privilege or copyright associated with the email or the attachments. If you are not the intended addressee, you must not use, transmit, disclose or copy the email or any attachments. If you receive this email by mistake, please notify the sender immediately and delete the original email. On 13 Jun 2015, at 6:49 am, Ursula Schulze-Gahmen uschulze-gah...@lbl.gov wrote: Does anyone have experience with Tris buffer in cryo protectants? I would expect the pH of the cryosolution to increase a lot during flash freezing which could perhaps destroy the diffraction. I rarely use Tris for crystallization but the current protein really prefers Tris. I would appreciate any comments. Ursula -- Ursula Schulze-Gahmen, Ph.D. Project Scientist UC Berkeley, QB3 360 Stanley Hall #3220 Berkeley, CA 94720-3220 (510) 643 9491
