Hi Andy,
I hope your offer for advice on crystallizing without O2 is still available. I am working on crystallizing some proteins that are oxygen-sensitive. We have a glove-box that I'm using and can make grid-screens using degassed solutions.
What kind of glovebox is it? There are several types, there are glove boxes which use N2 and keep a pressurein the glove box higher than the surrounding to keep oxygen out. and there are glove boxes http://www.coylab.com/vinylgb.htm), which use a mixture of N2/H2 at the same pressure as the surrounding and a catalyst that reduces O2 that diffusing into the glove box. I prefer the latter type; see below
However, I would like to use some of the available commercial sparse-matrix screens if possible. Is it possible to degas a large number of solutions (in a time-efficient way) to make this possible? Do you primarily degas solutions and then try to crystallize your samples in a glove-box?
We usually fill aliquots of 0.5-1 ml of crystallization screen solutions into 1.5 ml plastic screw cap vials (there are several types which have a rubber sealing). O2 can diffuse through plastic. Using screw cap vials prevents H2O to evaporate from your solutions. We keep those vials in the glove box for several weeks until usage. In the Coy type of glove box there is a steep gradient for O2 from the vial to the atmosphere of the glovebox, since O2 is consumed by reacton with H2 at the catalyst. We once measured the time how long it takes until the oxygen level in the solutions has dropped sufficiently but I forgot the exact numbers. 2-3 weeks should be fine. In the case of very sensitive proteins one can still add reducing agents. You should use sterile plastic vials or add azide to prevent microbial growth.
Is there a way to limit the oxygen availability inside sealed crystal trays that would allow putting them in a cold-room for example (we can't fit an incubator in our glovebox so crystallizing at different temperatures is currently not possible).
This is a bit tricky. We use plexiglass boxes which were made by the workshop here at the University. We prepare the crystallization plates in the glovebox and put them subsequently into the boxes, seal them and take them out for incubation at e.g. 4 deg C. Moreover, you can use plates with a sealing like the Qiagen/Nextal type and place them into the box. Then you have two barriers. You can put a vial into the box, that contains contains a solution reacting with oxygen like e.g dithionite or a solution of glucose oxidase/glucose consuming O2 that diffuses into the plexiglass box. With these boxes we can check the crystallization experiments under the microscope. However, any measure you take to keep oxygen out, after 2 weeks there will be already some oxygen. After 4 weeks even more. We followed the diffusion of oxygen into the plexiglass boxes using reduced methylviologen that is deep blue in the reduced state and reacts readily with oxygen. It works well for short time periods but is not suited for longer periods (1-3 months). For longer incubation periods and very sensitive proteins you have to crystallize in the glovebox. HTH Guenter
Thank you for your time, Best Regards, -Andy Torelli -----Original Message----- From: CCP4 bulletin board on behalf of Guenter Fritz Sent: Fri 9/26/2008 3:21 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] losing zinc during crystallization Hi Sue, I fully agree with Thierry, you might have to cyrstallize the protein under exclusion of dioxygen. There are many metallo proteins which have to be crystallized that way. But a first attempt might be the TCEP as already suggested by many others. If you need advice regarding crystallization without O2, send me a note. Good luck! Guenter
-- *********************************** Priv.Doz.Dr. Guenter Fritz Fachbereich Biologie Sektion Naturwissenschaften Universitaet Konstanz http://www.biologie.uni-konstanz.de/fritz Universitaetsstrasse 10 Postfach M665 D-78457 Konstanz e-mail: [EMAIL PROTECTED] Tel. Office: +49-(0)7531 88 3205 Tel. Lab : +49-(0)7531 88 3687 Fax: +49-(0)7531 88 2966
