July 20, 2010 - 1:10pm
I'm testing protocol 21.11.4 from Current Protocols in protein science. The denaturing buffer is said to contain 150 mM oxidized glutathione and 15 mM of reduced glutathione. In the past I've always used the reduced version 10X more than the oxidated. I would like to confirm those numbers. Thank you.
July 29, 2010 - 8:52am
#1
GSH:GSSH ratio
For oxidative folding the inclusion of GSH:GSSG (10:1) is a good starting point. In the following publication we used 5:1 and a very detailed protocol is described:
Wingfield et al (1999). “Functional and Biophysical Characterization of Full Length, Recombinant Human TIMP-2 Produced in Escherichia coli: Comparison of wild type and N-terminal Alanine substituted variant”. J. Biol. Chem. 274, 21362-21368.
Good luck
Paul
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July 29, 2010 - 2:26pm
#2
ratio of oxidized to reduced glutathione
Esmeralda:
The ration of oxidized to reduced glutathione is an experimental variable in refolding proteins. We usually do a series of experiments where the ratio is varied from 10-to-1 oxidized all the way to 1-to-10 oxidized to reduced. For the specific proteins described in the protocol you cite, the best results were obtained with the indicated ratio.
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