(1) Cleland, W.W. (1964) Biochemistry 3, 480-482
Dithiothreitol, a new protective reagent for SH groups.
(2) Zahler, W.L. & Cleland, W.W. (1968) J. Biol. Chem. 243, 716-719
A specific and sensitive assay for disulfides.
(3) Jocelyn, P.C. (1987) Methods Enzymol. 143, 246-256
Chemical reduction of disulfides.
(4) Gegenheimer, P. (1990) Methods Enzymol. 182, 174-193
Preparation of plant extracts.
(5) Hart, R.A. et al. (1994) Bio/Technology 12, 1113-1117
''Large scale in situ isolation'' o periplasmatic IGF-I from E. coli.
Dithiothreitol (DTT) is, like β-mercaptoethanol, a reducing reagent for proteins and protects the cysteine residues against oxidation. It may substitute for β-mercaptoethanol in almost all experiments at three to four fold lower concentrations. DTT is less toxic, its odor is less intensive and it doesn''t form mixed disulfides like β-mercaptoethanol. DTT is water-soluble and stock solutions are prepared at 1 M. Store the solution aliquoted at -20°C and protect from heat during the experiment. Do not choose a to low concentration for the experiment, because it is readily oxidized by air. The working concentration ranges from 0.1 to 1 mM, but the preparation of plant extracts ( 5 mM; ref. 4) or for the ''large scale in situ isolation'' of proteins after fermentation (10 mM; ref. 5) require higher concentrations. For the complete reduction of disulfides, the concentration might be significantly higher (3).
A more stable and odorless alternative to DTT is Tris(2-carboxy)ethylphosphine (A2233).