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REDUCTION BY DTT - (Jul/27/2003 )

I want to performe stability study of a protein using spectroscopic methods like CD, fluorescence. As the protein of interest have disulfide bond, I want to reduce it using DTT, so that we can understand the importance of the disuphide bond in stability of the protein. What is the usual protocol for reduction of protein for such studies? Will 10mM-20mM DTT and incubation at room tem for 2 hr / or overnight incubation at 4 degree, do the job? Does DTT interfare in spectroscopic techniques?
Thanking you
Biplab Bose
New Delhi


There is a product I have never used actually but which perhaps you may find interesting for reduction of disulphide bridges under gentle conditions: tris-2-carboxymethyl phosphine, or TCEP (Sigma cat no C4706 or Pierce cat no 20490).
I don't know if it has ver been used for your purpose, though.