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Hi, I am sorry that I cannot recall the highest concentration of DTT that I ever used (inadvertently) when working with disulphide-containing proteins. However I have the following mildly useful suggestion: If you have properly formed -S-S- as well as a bunch of free -SH perhaps a safer strategy is to modify the available -SH with something - it could be a reversible protective group or a permanent one such as iodiacetamide. This way you avoid playing with -S-S- shuffling entirely. Notably, this will reduce your chances of obaining a derivative since protected -SR are much less likely to react with heavy atom agents. As a useful bonus, if you modify the SH with e.g. iodoacetamide, then run MS on the intact protein - you will find out how many -SH remain unmodified. Among those, some are in -S-S- bonds and some are buried and therefore inaccessible to the reagent. By repeating iodoacetamide treatment post-reduction or on an unfolded sample you can further figure out the pattern. Good luck, Artem "Nothing is built on stone; all is built on sand, but we must build as if the sand were stone" Jorge Luis Borges _____ From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Brad Bennett Sent: Monday, August 10, 2009 5:06 PM To: [log in to unmask] Subject: [ccp4bb] Disulfide bond "survival" in the presence of DTT? Hi all- Got a perplexing thiol chemistry/phasing issue. To prevent non-native disulfide bonds from forming between free Cys but to preserve native disulfides, I have heard of using a very low (say < 0.5 mM) concentration of DTT. I've recently come across a paper where the assignment of 3 disulfides in a protein structure was carried out by calculating a 4A resolution anomalous difference map (detecting the sulfur anomalous signal with 1.7A x-rays). However, at every step in the protein prep and in the crystallization solution, there exists mM concentrations of DTT. Every protein is different, so the microenvironment and the protection of a disulfide will be different for every one you come across. But I am surprised that a disulfide can "withstand" that much DTT. I guess the proof is in the pudding, which would be the anomalous peaks observed in the maps. What is the upper limit on DTT (or B-ME or TCEP) concentration that people have used and still maintained native disulfides? Thanks- Brad