Which reducing reagents can be used to stabilize a thiol-dependent enzyme?
TCEP [tris-(2-carboxyethyl)phosphine] (PIERCE) can be used at 0.1-1 mM final concentration in column buffer to stabilize oxidation-sensitive proteins during purification [Burns, J.A., et. al., (1991) J. Org. Chem. 56, 2648-2650]. These compounds specifically reduce disulfide bonds without affecting intein-mediated cleavage reaction and thus it can be used to stabilize proteins with essential thiols. Furthermore, TCEP or TCCP (1-5 mM) may also be used as a reducing agent in SDS-PAGE Sample Buffer in place of DTT, which can cause partial or complete cleavage of the fusion protein when certain amino acids are at the position preceding the cleavage site (C-terminal fusion vectors).
