DL-Dithiothreitol (DTT) | CAS 3483-12-3 | ≥95%

Reducing agent for disulfide bonds in proteins — DTT is the standard small-molecule thiol reductant for cleaving disulfide bonds (S–S) in proteins and peptides. At concentrations of 1–10 mM, DTT quantitatively reduces intramolecular and intermolecular disulfide bridges to free thiols (–SH), denaturing protein tertiary structure and dissociating disulfide-linked subunits. This reaction is thermodynamically driven by the formation of a stable six-membered cyclic disulfide (oxidised DTT, trans-4,5-dihydroxy-1,2-dithiane), which makes DTT a more effective reductant than monothiols such as β-mercaptoethanol or glutathione. DTT was introduced by W. W. Cleland in 1964 and is frequently referred to as Cleland’s reagent (Cleland, Biochemistry 1964, 3, 480).

SDS-PAGE and protein electrophoresis — DTT is routinely included in Laemmli sample buffer at 50–100 mM to ensure complete reduction of disulfide bonds prior to denaturing polyacrylamide gel electrophoresis. Under these conditions, proteins migrate strictly according to molecular weight, unaffected by disulfide-mediated aggregation or conformational heterogeneity. DTT has largely replaced β-mercaptoethanol in many electrophoresis protocols due to its lower odour and more complete reduction at equivalent concentrations.

Protection of free thiols and enzyme active sites — DTT is added at 0.5–5 mM to protein purification buffers, enzyme assay mixtures, and cell lysis buffers to maintain cysteine residues in the reduced state and prevent oxidative inactivation of thiol-dependent enzymes. This is critical for cysteine proteases, protein tyrosine phosphatases, and other enzymes whose catalytic activity depends on a free thiol at the active site.

Protein refolding and disulfide shuffling — controlled ratios of reduced DTT and oxidised DTT (DTTox) are used to establish a redox buffer during in vitro protein refolding. By adjusting the DTT/DTTox ratio, researchers can promote the formation of native disulfide bonds while preventing kinetic trapping in non-native disulfide pairings. This approach is widely used in the refolding of recombinant proteins expressed as inclusion bodies in E. coli.

Restriction enzyme and ligase digestion buffers — many commercial restriction enzyme and DNA ligase reaction buffers contain 1–10 mM DTT to maintain enzyme stability and activity during molecular cloning workflows. DTT is a standard component of T4 DNA ligase buffer, T4 polynucleotide kinase buffer, and reverse transcriptase reaction mixes.

Cell biology and intracellular redox studies — DTT is used as a membrane-permeable reducing agent to induce endoplasmic reticulum (ER) stress by disrupting disulfide bond formation in the secretory pathway. At 1–5 mM, DTT triggers the unfolded protein response (UPR), making it a standard pharmacological tool for studying ER stress signalling and protein quality control mechanisms in mammalian cells.

Mass spectrometry sample preparation — in bottom-up proteomics, DTT reduction (typically 10 mM, 56 °C, 30 min) followed by iodoacetamide alkylation is the standard protocol for preparing protein digests prior to LC-MS/MS analysis. Complete disulfide reduction ensures that all cysteine-containing peptides are accessible for tryptic digestion and identified during database searching.

Thiol-reactive chemistry and bioconjugation — DTT is used to selectively reduce disulfide bonds in antibodies and other proteins to generate free thiols for subsequent conjugation with maleimide-functionalised payloads, fluorophores, or PEG chains. This approach is central to the preparation of antibody-drug conjugates and site-specifically labelled proteins for imaging and therapeutic applications.