Re: [ccp4bb] Reducing Agent Tips?

[Roger Rowlett]

Of these, typically TCEP>DTT>BME in terms of effectiveness in maintaining reduced Cys groups. Some proteins, when purified, require obscenely large reducing agent concentrations to keep them stable. One of our "horror show" proteins required 100 mM DTT + 10 uM EDTA to remain stable. You should consider including EDTA in the solution to sequester metal ions (if this is compatible with your protein). Metal ions, including the ubiquitious trace Zn2+, efficiently catalyze the oxidation of sulfhydryls. Include 1 uM or more if your protein will tolerate it. Cheers. Jeremiah Farelli wrote: Hello all, Anyone have any tips for reducing agents for use with the following crystallization conditions: 100 mM hepes, pH 7.5, 24% PEG 1500 or 100 mM Tris, pH 8.0, 24% PEG 1500 I've tried BME and DTT (1 mM). Currently trying out TCEP. The protein seems to be sensitive to oxidation, with a selenomet-derivative even more so. Thanks! -- Roger S. Rowlett Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu