As part of daily lab procedures, proteins and peptides need to be kept in their reduced state, e.g., for protein analysis, to maintain their activity, to prevent denaturation, to couple them to carriers or payloads, or to inactivate RNAses.

For this purpose, DTT and BME are frequently used, however, they come with certain drawbacks: DTT and BME are easily oxidized by ambient air, they may react with heavy metal ions and interfere with metal ion affinity chromatography (IMAC), they are quenching thiol-reactive reagents like maleimides, and, in the case of BME, it is malodorous.

In this blog, we present the reducing agent tris-(2-carboxyethyl)phosphine (TCEP) as nearly odorless alternative. You might already know TCEP as effective reagent for the cleavage of disulfide bonds, but there is more to discover!

Reduction of a disulfide with TCEP: The phosphine is oxidized to the corresponding phosphine oxide. Compared to DTT, TCEP’s reaction with the disulfide is irreversible.

In literature, TCEP is reported for various applications in protein science, e.g. reducing gel electrophoresis, immobilized metal ion affinity chromatography (IMAC), the derivatization of proteins with maleimide- or haloalkane-based linkers or for refolding proteins, also from inclusion bodies.

TCEP is stable in aqueous solutions, highly reactive, selective towards disulfide structures and - compared to DTT (RL-1020) - more resistant to oxidation by air. Its hydrochloride salt is readily soluble in aqueous media and a neutralized 0.5 M stock solution may be stored at -20 °C without degradation. For most applications, a final concentration of 0.1 mM - 1.0 mM is required. Moreover, TCEP can be applied at a wide pH range (1.5 - 8.5). Compared to DTT, it has shown to be significantly more stable at pH values above 7.5 and is a faster and stronger reductant at pH values below 8.0.

As TCEP does not contain thiols, excess reagent does not have to be removed, as it usually does not interfere with downstream processes. Compared to DTT, TCEP rather does not react with maleimides and is thus the preferred reagent for labeling cysteine residues with maleimides or haloalkanes.

And TCEP has even more advantages compared to DTT and BME: Is does not absorb UV light in the range between 250 - 285 nm, and thus does not interfere with photometric protein measurements. And, due to its charged nature, it does not permeate cell membranes.

Thus, when deciding which reducer to use, TCEP should be your reagent of choice! However, be careful with the abbreviation TCEP, which is also used for tris(2-chloroethyl)phosphate, a regulated plasticizer and flame retardant.

Besides, as a water-soluble Wittig reagent - TCEP is essentially a hydrophilic version of triphenylphosphine - it may be used together with a catalytic base (e.g., 10 mol% triethylamine) to link aldehyde functionalized biomolecules (accessible, e.g., by the Malaprade Oxidation of a terminal serine or threonine residue with periodate) with alkenes (like, e.g., maleimides as in SMCC, MAA1000, or Sulfo-SMCC, MAA1050), e.g., for attaching payloads in the creation of ADCs. When the pre-formed ylide from TCEP and the aldehyde is purified, disulfide bonds will not be significantly harmed, and thiols will not be alkylated.

→ Interested in maleimide-linkers for ADC construction? Browse our brochure Linkerology®

References:

A Comparison between the Sulfhydryl Reductants Tris(2-carboxyethyl)phosphine and Dithiothreitol for Use in Protein Biochemistry. E. Burmeister Getz, M. Xiao, T. Chakrabarty,R. Cooke, P. R. Selvin; Analytical Biochemistry 1999; 273: 73. https://doi.org/10.1006/abio.1999.4203

Molecular dissection of botulinum neurotoxin reveals interdomain chaperone function. A. Fischer, M. Montal; Toxicon 2013; 75: 101. https://doi.org/10.1016/j.toxicon.2013.01.007

Malaprade Reaction. Z. Wang. In Comprehensive Organic Name Reactions and Reagents; Z. Wang (Ed.).; 2010; https://doi.org/10.1002/9780470638859.conrr406

The Wittig bioconjugation of maleimide derived, water soluble phosphonium ylides to aldehyde-tagged proteins. Rafael W. Hartmann, Matthijs Pijnappel, Johan Nilvebrant, Hildur Run Helgudottir, A. Asbjarnarson, G. Asta Traustadottir, T. Gudjonsson, P.-Å. Nygren, F. Lehmann, L. R. Odell; Org. Biomol. Chem. 2021; 19: 10417. https://doi.org/10.1039/D1OB01155C

Investigation of protein refolding using a fractional factorial screen: A study of reagent effects and interactions. M. Swope Willis, J. K. Hogan, P. Prabhakar, X. Liu, K. Tsai, Y. Wei, T. Fox; Prot. Sci. 2005, 14: 1818. https://doi.org/10.1110/ps.051433205

Alternative preparation of inclusion bodies excludes interfering non-protein contaminants and improves the yield of recombinant proinsulin. R.B. Mackin; MethodsX 2014, 1: 108. https://doi.org/10.1016/j.mex.2014.07.005