Effect of reducing agent added to Tris buffer

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Trimethylolaminomethane Tris buffer has good compatibility with nucleic acid and protein, and is widely used in various biological experiments. Tris has many buffer solution systems, such as Tris hydrochloric acid, Tris acetic acid, TAE, tbe, etc. many buffers also add reducing agents such as DTT, mercaptoethanol or TCEP. What is the role

Effect of reducing agent added to Tris buffer

Reducing agent is added to Tris buffer to protect the stability of protein. It is usually used for protein separation and purification, Western blot, protein identification and other experiments. Proteins are composed of dehydration condensation of a variety of amino acids, some may contain cysteine residues, which are easy to be oxidized and may lead to protein aggregation and precipitation. Therefore, add reducing agent DTT, TCEP or mercaptoethanol to Tris buffer to prevent the above problems.

Three reducing agents in Tris buffer

1. Dithiothreitol, referred to as DTT for short, is usually added to the buffer in the process of protein purification. It is a common small molecule organic reducing agent with antioxidant effect. The chemical formula is c4h10o2s2, which is a linear molecule in the reduced state and becomes a six membered ring structure containing disulfide bond after oxidation. The addition concentration is 5 ~ 10mm, and the reductant concentration needs to be much higher than the protein concentration in the buffer.

2. Tris (2-carboxyethyl) phosphine, referred to as TCEP, is a very effective mercaptan reducing agent. It is widely used as a quantitative reducing agent of disulfide bond in protein chemistry and proteomics. Compared with other thiol reducing agents such as DTT, TCEP is not only an efficient disulfide bond reducing agent, but also does not need to be removed in the crosslinking reaction of some sulfhydryl groups. TCEP is more stable but more expensive.

3. Mercaptoethanol, i.e β- Mercaptoethanol (BME) can protect free sulfhydryl groups on proteins from oxidation, so as to avoid protein aggregation or denaturation. It has only one sulfhydryl group and poor reduction ability. It should be added every two or three days to ensure the effect. Because it is volatile, beta mercaptoethanol can be added to the pool liquid and diffused into the droplets by its volatilization.

Tris buffer is usually prepared at room temperature, while reducing agent DTT and mercaptoethanol will degrade at room temperature. If reducing agent is added to the buffer, it needs to be refrigerated at low temperature, or add reducing agent to Tris when in use. Desheng has rich production and R & D experience in biological buffer, and can provide Tris, mops, HEPES and other reagents.