Instructions for use

TCEP hydrochloride is an effective, universal, odorless, and thiol free reducing agent widely used in protein and other studies involving 

disulfide bond reduction (Figure 1). This product has good solubility and stability in many solvents, and the effect of reducing disulfide 

bonds is similar to DTT, but unlike DTT or other sulfur-containing reducing agents that must be removed before the thiol crosslinking reaction.

The ability and advantages of trialkyl phosphine compounds to reduce protein disulfide bonds have long been well-known [1] [2], but their 

application is limited due to their poor solubility in water and unpleasant odor. Phosphates are relatively stable in aqueous solutions, can selectively

 cleave disulfide bonds, and have almost no effect on other functional groups [2]. The discovery of tris (2-carboxyethyl) phosphine (TCEP) overcomes 

the shortcomings of trialkyl phosphine compounds and is widely used [3].

TCEP can selectively and thoroughly reduce stable alkyl disulfide compounds in water over a wide pH range [4], with reduction occurring at room 

temperature for less than 5 minutes. TCEP has no volatile or irritating odor, and is not easily oxidized in the air like most other reducing agents. 

Compared with DTT, TCEP is more stable, more active, and can reduce disulfide bonds at low pH values [5].

Product Information

1. Solubility

TCEP hydrochloride (molecular weight: 286.65) has good solubility in water, up to 310g/L (1.08M). As a hydrophilic compound, TCEP has excellent 

solubility at almost any pH in water buffer, making it easy to prepare reaction concentrations and 10X storage solutions in most water buffer solutions. 

The only solvents with lower solubility of TCEP in organic solvents are methanol and ethanol.

2. Stability in solution

TCEP is very stable in water, acid, and ordinary solutions, with a pH of approximately 2.5 when dissolved in water. Research has shown that TCEP 

remains unchanged in 100mM HCl or 100mM NaOH solution at room temperature for 24 hours, and also remains unchanged in the following 50mM 

buffer solutions: Tris • HCl (pH 7.5, 8.5, and 9.5), HEPES (pH 6.8 and 8.2), borate buffer solutions (pH 8.2 and 10.2), and CAPS (pH 9.7 and 11.1) [5]. 

Even after three weeks in these buffer solutions, the oxidized portion of TCEP is less than 20%.

TCEP is not particularly stable in phosphate buffer, especially under neutral pH conditions. The experiment showed that it was completely oxidized 

within 72 hours in 0.35M phosphate buffer salt (PBS) with a pH of 7.0, which is the same as about 50% of the oxidation time in 0.15M PBS with a pH 

of 8.0. TCEP only undergoes slight oxidation in PBS with pH>10.5 or<6.0. Therefore, if TCEP is used in PBS buffer, it is best to prepare and use it immediately.

3. pH range of reducing activity

TCEP can effectively reduce disulfide bonds over a wide pH range [5]. Experiments have shown that TCEP can completely reduce 

2,2 '- dithiodipyridine (2,2' - DTDP) within 30 seconds between 1.5<pH<9.0. However, at pH>9, only 50% of 2,2 '- DTDP is reduced. At pH<8.0, TCEP 

performs better than DTT and can even reduce oxidized DTT [5].

4. Reaction concentration

For most reactions, 5-50mM TCEP at room temperature can provide sufficient molar concentration to effectively reduce disulfide bonds of peptides or 

proteins within a few minutes. When the pH is between 1.5 and 8.5, a 30 µ M TCEP can completely reduce 2,2 '- DTDP (20 µ M) within 40 seconds [5]. 

When using an equal molar amount of TCEP, the reaction time will be much longer. The Mery report shows that when the molar ratio of TCEP to disulfide 

compound is 1:1, the reaction time is nearly one hour [6].

5. Use compatibility

Because TCEP does not contain thiols, it does not need to be removed before labeling or crosslinking with maleimide. In most cases, when the TCEP 

concentration is less than 10-20MM, the chemical reaction with maleimide is also compatible.

TCEP (50mM) can replace DTT or 2-mercaptoethanol as a reducing sample buffer for SDSPAGE. However, because TCEP carries a charge in the solution, 

it is not suitable for two-dimensional isoelectric focusing electrophoresis (IEF).

One strategy for labeling antibodies is to reduce the disulfide bonds in the hinge region of IgG molecules without the need for additional labeling to 

reduce the connection between heavy and light chains. This "partial" reduction is typically achieved using 2-mercaptoethylamine (2-MEA). Bl ä uenstein 

et al. [7] demonstrated that TCEP has the following effect: adding TCEP with a final concentration of 3.8-4.0mM to 0.1M phosphate buffer containing 

10mg/mL IgG (pH 4.6-7.5), reacting at room temperature for 20-30 minutes, and then removing TCEP products using a desalination column.

Reference:

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

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

5. 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. 

6. 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. 

7. Blauenstein, P., et al