DTT, short for dithiothreitol and also known as Cleland’s reagent, is one of the most common reducing agents in biochemistry. If you have run an SDS-PAGE gel, purified a protein or set up an enzyme assay, you have probably used it. This guide explains what DTT does, how it is used, how to store it and when to choose an alternative.
Key facts
| Name | Dithiothreitol (DTT), Cleland’s reagent |
|---|---|
| Formula | C₄H₁₀O₂S₂ |
| Molar mass | 154.25 g/mol |
| CAS number | 3483-12-3 (DL-form, the usual commercial product) |
| Appearance | White solid with a mild sulfur odour |
| Role | Reducing agent for disulfide bonds |
What DTT does
Proteins often contain disulfide bonds, S–S bridges between cysteine residues. DTT has two thiol (–SH) groups. It reduces a disulfide to two free thiols, and in the process DTT itself closes into a stable six-membered ring. That ring formation drives the reaction forward, which is why DTT is so effective at low concentrations.
Where DTT is used
- SDS-PAGE sample buffer: to break disulfide bonds so proteins separate by size.
- Protein purification and storage: to keep cysteines reduced and prevent unwanted aggregation.
- Enzyme assays: to protect enzymes that need free thiols.
- Molecular biology: in some reverse transcription and other enzyme buffers.
- Immunoassays: in sample treatment steps alongside detection reagents such as HRP-conjugated secondary antibodies (for example anti-mouse HRP). Note that strong reducing conditions can affect antibodies themselves, so follow the kit protocol.
Practical tips
- Make solutions fresh or store aliquots frozen; DTT oxidises in air, faster at higher pH and in the presence of metal ions.
- Add it late to buffers that will sit for a long time.
- Check compatibility: DTT interferes with some protein assays, metal-affinity columns (Ni-NTA) and maleimide labelling chemistry.
- Handle safely: DTT is harmful if swallowed and irritating to skin and eyes. Wear gloves and eye protection.
Alternatives to DTT
| Reagent | Advantages | Drawbacks |
|---|---|---|
| TCEP | Odourless, stable in air, works over a wide pH range, compatible with Ni-NTA | Can be more expensive; acidic stock needs neutralising |
| β-Mercaptoethanol | Cheap and widely used | Strong odour, volatile, needed at higher concentrations |
| DTE (dithioerythritol) | Isomer of DTT with similar behaviour | Less common |
Buying DTT
Molecular biology grade DTT is tested for nucleases and proteases; standard grades suit general chemistry. Buy small packs, store dry and cold, and search by CAS number to avoid confusion with DTE. See also our guide to reagent grades.
Frequently asked questions
What concentration of DTT is typical?
It depends on the application and protocol; sample buffers usually use more than storage buffers. Follow your method or kit instructions.
Why does my DTT stock smell?
A faint sulfur smell is normal. A strong smell or yellowing suggests degradation, so make a fresh stock.
Is DTT the same as Cleland’s reagent?
Yes. It is named after W. Wallace Cleland, who introduced it.
