Reduction of Disulfide bonds using Bondbreaker TCEP Solution.

Bond-Breaker TCEP Solution, is a potent, odorless, thiol-free reducing agent with broad application to protein and other research involving reduction of disulfide bonds. This product is an effective and convenient replacement for B-mercaptoethanol or DTT in SDS-PAGE sample buffers. The neutral pH of this reagent provides sharp bands and avoids exposing proteins to the strong acid of TCEP•HCl, which can result in hydrolysis and carbohydrate modification.

Protocol for using Bond-Breaker TCEP Solution to reduce proteins for SDS-PAGE analysis.

Advantages of Bondbreaker TCEP for reducing disulfides in peptides and proteins:

• Odorless – Unlike DTT or BME, TCEP is odor-free
• Stable in air – The inherent stability of the TCEP moiety eliminates the need for any special precautions to avoid oxidation of the 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.

Bondbreaker TCEP Highlights:

• Odorless – contributes to a healthier lab environment
• Specific – selective and complete reduction of even the most stable water-soluble alkyl 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
• Versatile – reduces peptides and proteins over a broad range of pH, salt, detergent and temperature conditions
• Efficient – reverses disulfide bonds; excellent alternative for uncoupling crosslinked proteins or peptides by agents such as DSP and DTSSP.

Considerations for use of Bondbreaker TCEP

• 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 TCEP solution as this will decrease TCEP 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
TCEP•HCl
Ellman’s Reagent
Sulfhydryl-reactive Biotinylation Reagents
Sulfhydryl-reactive Crosslinking Reagents
SulfoLink Coupling Gel (Sulfhydryl immobilization)
Maleimide-activated Carrier Proteins

References

1. Irsch, T. and Krauth-Siegel, R.L. (2004) Glyoxalase II of African Trypanosomes Is Trypanothione-dependent. J. Biol. Chem.279, 22209-22217.
2. Schmidt, H. and Krauth-Siegel, R.L. (2003) Functional and Physicochemical Characterization of the Thioredoxin System in Trypanosoma brucei. J. Biol. Chem.278, 46329-46336.