Thermo Scientific Bond-Breaker TCEP Solution, Neutral pH is a stable, 0.5M solution of the thiol-free, phosphine-based TCEP compound, useful as a 10X stock for addition to SDS-PAGE sample loading buffers to reduce protein disulfide bonds.
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.
- 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.
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 to 50mM TCEP provides sufficient molar excess to effectively reduce peptide or protein disulfide bonds within a few minutes at room temperature.
|Protocol for using Thermo Scientific Bond-Breaker TCEP Solution to reduce proteins for SDS-PAGE analysis.
|Properties of Bondbreaker TCEP
||6.6 + 0.1
||Store at room temperature.
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.
- Irsch, T. and Krauth-Siegel, R.L. (2004) Glyoxalase II of African Trypanosomes Is Trypanothione-dependent. J. Biol. Chem.279, 22209-22217.
- 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.
Immobilized TCEP Disulfide Reducing Gel
DTT and other Reducing Agents