The ability of trialkylphosphine compounds to reduce protein disulfide bonds have been known for many years.^1,2 Phosphines are stable in aqueous solution, selectively reduce disulfide bonds, and are essentially nonreactive toward other functional groups commonly present in proteins.² Trialkylphosphines, however, were hindered by their instability in water and their disagreeable odor. These obstacles were overcome by discovery of tris(2-carboxyethyl)phosphine (TCEP).^3-17

Tris[2-carboxyethyl] phosphine (TCEP) is a popular odorless reducing agent for protein applications. The uses for TCEP have recently been expanded with the introduction of a stable, neutral pH TCEP solution, Bond-Breaker TCEP Solution, that is ideal for preparing proteins for SDS-PAGE analysis. Reducing peptides and proteins without contaminating the sample with reducing agent is now possible with Immobilized TCEP Gel where TCEP has been covalently attached to an agarose support.

TCEP selectively and completely reduces even the most stable water-soluble alkyl disulfides over a wide pH range. Reductions frequently require less than 5 minutes at room temperature. TCEP is non-volatile, odorless, and unlike most other reducing agents, is resistant to air oxidation. Compared to DTT, TCEP is more stable, more effective, and able to reduce disulfide bonds at lower pHs.

Reduction of Disulfide bonds using Bondbreaker TCEP Solution.

Advantages of TCEP·HCl over traditional alternatives for reducing disulfides:

• Odorless – Unlike DTT or BME, TCEP is odor-free, so reductions can be carried out conveniently on the bench top.
• Stable in air – The inherent stability of the TCEP moiety eliminates the need for any special precautions to avoid oxidation when handling, using or storing TCEP.
• Efficient – For most applications, 5-50 mM TCEP provides sufficient molar excess to effectively reduce peptide or protein disulfide bonds within a few minutes at room temperature.
• Compatible – With TCEP, removal of the reducing agent is not necessary prior to most applications, (e.g. histidine-tagged protein purification, maleimide conjugations).

TCEP·HCl Highlights:

• Odorless – reduce proteins at your desktop; contributes to a healthier lab environment
• Specific – selective and complete reduction of even the most stable water-soluble disulfides
• Simple – effective reduction at room temperature and pH 5 in less than five minutes
• Stable – resistant to air oxidation; nonvolatile and nonreactive toward other functional groups found in proteins

Considerations for use of TCEP·HCl

• TCEP is generally very soluble in aqueous buffers at nearly any pH. Therefore, working concentrations and 10X stock solutions may be readily prepared in most aqueous buffers
• TCEP is stable in aqueous, acidic, and basic solutions. When TCEP is dissolved directly in water, the resulting pH is approximately 2.5.
• TCEP is not particularly stable in phosphate buffers, especially at neutral pH. Therefore, if TCEP is to be used in PBS buffers, prepare the working solution immediately before use.
• TCEP may be used as a substitute for DTT or 2-mercaptoethanol (2-ME) in sample loading buffer for SDS-PAGE; use a final concentration of 50 mM TCEP.
• Because TCEP is charged in solution, it is not compatible for use in isoelectric focusing.

Related pages
Immobilized TCEP Disulfide Reducing Gel
Bondbreaker TCEP Solution
Ellman’s Reagent
Sulfhydryl-reactive Biotinylation Reagents
Sulfhydryl-reactive Crosslinking Reagents
SulfoLink Coupling Gel (Sulfhydryl immobilization)
Maleimide-activated Carrier Proteins

References

1. Ruegg, U.T and Rudinger, J. (1977). Reductive cleavage of cystine disulfides with tributylphosphine. Methods Enzymol. 47, 111-126.
2. Kirley, T.L. (1989). Reduction and fluorescent labeling of cyst(e)ine-containing proteins for subsequent structural analysis. Anal. Biochem. 180, 231.
3. Burns, J.A., et al. (1991). Selective reduction of disulfides by tris-(2-carboxyethyl)-phosphine. J. Org. Chem. 56, 2648-2650.
4. Han, J., et.al. (1999). Tris[2-carboxyethyl]phosphine – A reducing agent with versatile applications including cleavage of disulfide bonds and quantitation of numerous oxidants. Previews 2(4), 16-21.
5. Han, J.C. and Han, G.Y. (1994). A procedure for quantitative determination of tris(2-carboxyethyl)phosphine, an odorless reducing agent more stable and effective than dithiothreitol. Anal. Biochem. 220, 5-10.
6. Mery, J., et al. (1993). Disulfide linkage to polyacrylic resin for automated Fmoc peptide synthesis, immunochemical applications of peptide resin and mercaptoamide peptide. Int. J. Pept. Protein Res. 42, 44-52.
7. Gray, W.R. (1993) Disulfide structures of highly bridged peptides: a new strategy for analysis. Protein Sci. 2, 1732-1748.
8. Fisher, W.H., et al. (1993) In situ reduction suitable for matrix-assisted laser desorption/ionization and liquid secondary ionization using tris(2- carboxyethyl)phosphine. Rapid Commun. Mass Spectrom. 7, 225-228.
9. Bieri, S., et al. (1995) Disulfide bridges of a cysteine-rich repeat of the LDL receptor ligand-binding domain. Biochemistry 34, 13059-13065.
10. Tam, J.P., et al. (1995) Peptide synthesis using unprotected peptides through orthogonal coupling methods. Proc. Natl. Acad. Sci. USA 92, 12485-12489.
11. Blauenstein, P., et al. (1995) Experience with the iodine-123 and technetium-99m labelled anti-granulocyte antibody MAb47: a comparison of labelling methods. Eur. J. Nucl. Med. 22, 690-698.
12. Gorman, J.J., et al. (1996) Use of 2,6-dihydroxyacetophenone for analysis of fragile peptides, disulphide bonding and small proteins by matrix assisted laser desorption/ionization. Rapid Commun. Mass Spectrom. 10, 529-536.
13. Kirsch, T., et al. (1996) Cloning, high-yield expression in Escherichia coli, and purification of biologically active HIV-1 Tat protein. Protein Express. Purif. 8, 75-84.
14. Wu, J. and Watson, J.T. (1997) A novel methodology for assignment of disulfide bond pairings in proteins. Protein Sci. 6, 391-398.
15. Bernard, C.L., et al. (1997) Exp. Brain Res. 113, 343-352.
16. Huh , K. and Wenthold, R.J. (1999) J. Biol. Chem.274, 151-157.
17. Oda , Y., et al. (2001). Nature Biotech19, 379-382.

* TCEP·HCl is available in bulk quantities for manufacturing applications.