I have a protein which I know is stable with the presence of co-factors (NAD+ and Fe2+). Recently i have been trying to purify it with the TCEP as a reducing agent since my protein is known to have 5 cysteins, whereby 2 cysteins are exposed to the surface. I intend to try and crystallise this protein with the presence of the co-factors and TCEP. Is this possible? I've known that TCEP isn't volatile and thus needs to be added to the protein right before it is subjected to crystallisation and my concern is, is TCEP, and the cofactors compatible? Any tips on crystallising with cofactors and reducing agents?
I think you are going to have a problem keeping the Fe2+ reduced in the presence of air. Then, once it is oxidized to Fe3+, it will probably catalyze oxidation of TCEP and sulfhydryls. You may have to do your crystallization under an oxygen-depleted inert atmosphere. I suggest you consult literature on crystallization of proteins containing Fe2+ or FeS cofactors.
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