Tag Archives: protein reduction

TCEP or DTT?

Which reagent is better for reducing disulfides, DTT or TCEP? The two reagents are quite different in their reactivity, stability towards oxidation, reaction mechanisms, and other categories.

DTT and TCEP

DTT is a thiol-containing reagent, and this must be considered in applications involving thiol labeling. TCEP is charged in solution and should not be used in isoelectric focusing. Aqueous solutions of TCEP are quite acidic (pH 2-3).

TCEP HCl is odorless, air-stable crystalline solid, soluble in water at a > 1 M concentrations. It reduces disulfides at room temperature in < 5 min in dilute solutions (5 -50 mM). There is no need to remove TCEP prior to the use of sulfhydryl-reactive labels or crosslinkers. TCEP is selective toward disulfides, and is reactive at a broad pH range.

For those who love Chemistry:

TCEP mechanism

Reduction of disulfide with TCEP. First step is rate-determining, kinetic rather than thermodynamic control.

 

Rxn DTT

Reduction of disulfide with DTT. Formation of stable cyclic disulfide drives the reaction.

You can learn more about DTT and TCEP from this article: “A Comparison between the Sulfhydryl Reductants Tris(2-carboxyethyl)phosphine and Dithiothreitol for Use in Protein Biochemistry”, Elise Burmeister Getz et al.  Analytical Biochemistry 273, p. 73–80 (1999)