Structure of TCEP (Tris(2-carboxyethyl)phosphine hydrochloride).

Trialkylphosphines serve as excellent agents for the reduction of disulfide bonds in proteins, peptides and other disulfide bond-containing molecules^1,2 and are relatively unreactive toward other functional groups.³ The trialkylphosphine TCEP is stable in aqueous solutions and does not undergo the rapid oxidation that often occurs with other reducing agents such as dithiotreitol (DTT) and β-mercaptoethanol (BME).⁴ TCEP does not interfere with common sulfhydryl-reactive reagents (e.g., maleimide crosslinkers).⁵ Nevertheless, many protocols require recovery of the reduced sample separate from the reducing agent. Immobilized TCEP Disulfide Reducing Gel eliminates the need to use laborious and inefficient gel filtration methods to separate the reduced sample from the reducing agent. Immobilized TCEP Disulfide Reducing Gel may be adapted conveniently to a variety of scales and formats (i.e. batch, spin column and gravity-flow column procedures).

Advantages over traditional soluble alternatives for disulfide reduction of peptides and proteins:

• Excellent recovery free from reducing agent – Removing the reducing agent and recovering the reduced molecule without sample loss is a problem inherent to DTT or ß-mercaptoethanol (BME). Immobilized TCEP allows you to recover reduced protein/peptide in high yield (85% or higher) without the need to dialyze or desalt.
• Odorless – Unlike DTT or BME, Immobilized TCEP is odor-free, so reductions can be carried out conveniently on the bench top.
• Stable in air – The inherent stability of TCEP eliminates any need for special precautions to avoid oxidation when handling, using or storing Immobilized TCEP Disulfide Reducing Gel.
• Ease of handling – Immobilized TCEP reductant, allows you to easily dispense the amount of support required for each application. Reductions can be conducted over a broad range of pH values (pH 4.0-9.0) and temperatures (5°C-95°C).

Immobilized TCEP efficiently reduces antibodies from various species. Human, rabbit, mouse and goat IgG, each at a concentration of 100 µg/ml, were either directly loaded onto a 4-20% SDS-PAGE gel or treated with Immobilized TCEP in Tris buffer, pH 7.2 for 60 minutes at room temperature before loading onto the gel (2.8 µg of each sample, non-reduced and reduced were loaded per well). Lane1: Molecular Weight Marker, Lane2: Human IgG (non-reduced), Lane3 : Human IgG (reduced) , Lane4: Rabbit IgG (non-reduced), Lane 5: Rabbit IgG (reduced), Lane 6: Mouse IgG ( n o n - reduced), Lane 7: Mouse IgG (reduced), Lane 8: Goat IgG (non-reduced), and Lane 9: Goat IgG (reduced).

Immobilized TCEP Highlights:

• Odorless – reduce proteins at your desktop; contributes to a healthier lab environment
• Stable – virtually no loss of activity when stored refrigerated
• Versatile – reduces peptides and proteins over a broad range of pH, salt, detergent and temperature conditions
• Flexible – resin slurry provides flexibility for use in spin tube, column or batch modes
• Time-saving – no soluble reductant removal saves time and conserves sample
• Efficient – reverses both natural disulfide bonds and disulfide bonds artificially incorporated by crosslinking; excellent alternative for uncoupling crosslinked proteins or peptides by agents such as DSP and DTSSP

Properties of Immobilized TCEP Disulfide Reducing Gel 

Considerations for use of Immobilized TCEP Disulfide Reducing Gel 

• Reduction occurs over a wide range of pH (pH 4.0-9.0) and temperature (5°-95°C).
• Most proteins are reduced efficiently without a denaturant. However, adding a denaturant such as guanidine•HCl may aid in exposing internal disulfides to the Immobilized TCEP.
• Urea is not recommended as a denaturant as it forms cyanates that react with sulfhydryl groups.
• Do not allow metals to contact the Immobilized TCEP as this will decrease its activity.
• Including 5–20 mM EDTA in the sample buffer during reduction helps prevent reoxidation of the sulfhydryl groups by divalent metals such as Zn 2+, Cu 2+ and Mg 2+.
• The reduced sample should be used immediately after reduction because disulfides will reform over time.

Related pages
Immobilized TCEP Disulfide Reducing Gel – FAQ
TCEP•HCl
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, 111-26.
2. Kirley, T.L. (1989. Reduction and fluorescent labeling of cyst(e)ine-containing proteins for subsequent structural analysis. Anal. Biochem. 180, 231.
3. Levison , M.E. , et al. (1969) Reduction of biological substances by water-soluble phosphines: Gamma-globulin. Experentia 25, 126-7.
4. Han, J.C., et al. (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.
5. Haugland, R.P. and Blalgat, M.K. (1998). Preparation of Avidin Conjugates. In J.D. Pound (ed.) Immunological Protocols, Humana Press Inc., Totowa, NJ, 80, 189-90.