TCEP Operation Instruction

Product Description

Product name: TCEP-HCL

CAS NO. 51805-45-9

Product No. BF-02

Appearance: White crystal

Introduction: TCEP-HCL is a thiol-free and odorless reducing agent used in proteins, peptides, and other systems involving disulfide bond reduction. In clinical tests, TCEP-HCL is mainly used in homocysteine assay kits as a reducing agent to convert oxidized homocysteine to free homocysteine. Advantages: 1, No odor, non-voltile, anti-air oxidation. 2, Reducibility can be maintained for 2-3 weeks. 3. Not affected by metal ions, suitable for Ni2+ column purification of His-tagged proteins.

Usage: IVD—Test Kits Biochemistry–SDS PAGE, immobilized metal ion affinity chromatography (IMAC) experiments.

Storage

2-8°C, keep in dry and sealed container, avoid sunlight

Operation Instruction

TCEP hydrochloride is an effective, general-purpose, odorless, mercaptan-free reducer which is widely used in protein and other studies involving the reduction of disulfide bonds. This reducer has good solubility and stability in many solvents. The effect of reducing disulfide bonds is similar to that of DTT, but unlike DTT or other sulfur-containing reducing agents, which must be removed prior to the sulfhydryl cross-linking reaction.

Trialkylphosphine compounds have long been known for their ability and advantages to reduce protein disulfide bonds, but have been less widely used due to their poor solubility in water and unpleasant odor.Phosphates are stable in aqueous solution, can selectively break disulfide bonds, and have little effect on other functional groups. The discovery of tris (2-carboxyethyl) phosphine (TCEP) overcomes the shortcomings of trialkyl phosphine compounds and has been widely used.

TCEP can selectively and completely reduce alkyl disulfide compounds in water over a wide pH range, and the reduction can even take less than 5 minutes at room temperature.TCEP is not volatile, has no irritating odor, and is not susceptible to oxidation in the air like most other reducing agents.Compared with DTT, TCEP is more stable, more reactive, and can reduce disulfide bonds at low pH.

More information about TCEP

1, Solubility

TCEP hydrochloride (molecular weight: 286.65) has good solubility in water, up to 310g/L (1.08M).As a hydrophilic compound, TCEP is very soluble in water buffers at almost any pH, so it is easy to prepare reaction concentrations and 10X storage solutions in most water buffers.The only organic solvents with low solubility of TCEP are methanol and ethanol.

2, Stability

TCEP is very stable in water, acid, and ordinary solutions and is soluble in water at a pH of about 2.5.Studies have shown that TCEP does not change when being placed in 100mM HCl or 100mM NaOH solution for 24 hours at room temperature, nor does it change in the following 50mM buffer solution:Tris•HCl (pH 7.5, 8.5, and 9.5), HEPES (pH6.8, and 8.2), borate buffers (pH8.2, and 10.2), and CAPS (pH9.7, and 11.1), and even TCEP oxidized part is less than 20% in these buffers for three weeks

TCEP is not particularly stable in phosphate buffers, especially at neutral pH.The experiment showed that the total oxidation time in 0.35M phosphate buffer salt (PBS) at pH 7.0 was within 72 hours, the same as that in 0.15M PBS at pH 8.0 for about 50% oxidation time. It is only in pH＞10.5 or＜ 6.0 PBS, only slight oxidation occurred in TCEP.Therefore, if TCEP is used in a PBS buffer, it is best to use it on the spot.

3, pH Range

TCEP can effectively reduce disulfide bonds over a wide pH range, and the experimental results show that TCEP reduces disulfide bonds at 1.5＜pH＜9.0, 2,2 ‘dithiodipyridine (2,2’ -DTDP) was completely reduced within 30 seconds, but only 50% of 2,2 ‘-DTDP was reduced. TCEP is better than DTT and can even reduce oxidized DTT at pH＜8.0.

4, Concentration

For most reactions, 5-50mM TCEP at room temperature can provide sufficient molar concentration to effectively reduce the disulfide bonds of peptides or proteins in a matter of minutes. At pH 1.5-8.5, 2,2 ‘-DTDP(20 μM) can be completely reduced in 40s at 30 μM TCEP.When TCEP with equal molar amount is used, the reaction time will be much longer. The Mery report shows that when the molar ratio of TCEP and disulfide compound is 1:1, the reaction time will be nearly one hour.

5, Compatibility

Because TCEP does not contain mercaptan, removal is not required prior to maleimide labeling or cross-linking reactions. In most cases, chemical reactions with maleimides are compatible with TCEP concentrations less than 10-20mM.

TCEP (50mM) can be used as an alternative to DTT or 2-mercaptoethanol as a reduction sample buffer for SDSPAGE. However, TCEP is not suitable for two-dimensional isoelectric focusing electrophoresis (IEF) because of its charge in solution.

One strategy for labeling antibodies is to reduce disulfide bonds in the hinge region of the IgG molecule without the need for additional markers to reduce the connections between heavy and light chains. This “partial” reduction is usually accomplished with 2-mercaptoethylamine (2-MEA).Blauenstein demonstrated the effect of TCEP by adding a final concentration of 3.8-4.0mM to a 0.1M phosphate buffer containing 10mg/mL IgG (pH 4.6-7.5) at room temperature for 20-30 minutes, and then removing the TCEP product using a desalination column.

Reference:

Ruegg, U.T. and Rudinger, J. (1977). Reductive cleavage of cystine disulfides with tributylphosphine. Methods Enzymol 47:111-26.

Kirley, T.L. (1989). Reduction and fluorescent labeling of cyst(e)ine-containing proteins for subsequent structural analysis. Anal Biochem 180:231.

Burns, J.A., et al. (1991). Selective reduction of disulfides by tris-(2-carboxyethyl)-phosphine. J Org Chem 56:2648-50.

Levison, M.E., et al. (1969). Reduction of biological substances by water-soluble phosphines: Gamma-globulin. Experentia 25:126-7.

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.

Mery, J., et al. (1993). Disulfide linkage to polyacrylic resin for automated Fmoc peptide synthesis. Immunochemical applications of peptide resins and mercaptoamide peptides. Int J Peptide Protein Res 42:44-52.

Blauenstein, P., et al. (1995). Experience with the iodine-123 and technetium-99m labeled anti-granulocyte antibody Mab47: a comparison of labeling methods. Eur J Nuclear Med 22:690-8.