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Re: [ccp4bb]: preventing oxidation of Selenium

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I just wanted to thank you for your reply. Everything you wrote was very
useful to me, I appreciate it. I have gotten xtals with the SeMet and I am
just keeping my fingers crossed that everything is ok.

Thanks again,

On Thu, 16 Mar 2000, Frank von Delft wrote:

> >       I apologize that this is not a ccp4 question, but I know many of
> > you have experience with selenium-derivatized proteins and was curious to 
> > know if there was a consensus on what strategy is best to use to prevent
> > oxidation of seleno-methionine.	
> > 
> >       I will be obtaining a 40mer peptide which oligomerizes and has 1
> > selenium/chain. I was wondering if anyone could offer any tips on
> > preventing oxidation:  is it enough just to add 10mM DTT or considering
> > this is a peptide is it better to use a glove bag purged with nitrogen? 
> > Also, how long can a SeMet crystal be manipulated while exposed to air?
> In one case, I added DTT to the crystallisation conditions and 10 days
> later cryo-stored the xtals.  From the fluorescence spectrum it looked
> like there was significant oxidation. 
> In the next case, I could not add DTT if I wanted decent crystals, so I
> just left it away completely, and the xtals were exposed to oxygen for
> some 3 days. But during cryoprotection, about 1 hour before freezing, I
> added 3mM DTT to the buffer.  And these crystal seemed nearly completely
> non-oxidised, with a proper sharp peak in the fluorescence spectrum.
> It seems that oxidation is reversible, so you may find this second
> treatment adequate, probably in fact more reliable, because DTT goes off
> quite quickly, but oxygen diffuses through the Linbro plate plastic (I
> have been told).  
> Cheers 
> Phraenquex