2-Mercaptoethylamine reduction

To each ml of the antibody solution, add 6 mg of 2-mercaptoethylamine hydrochloride (final concentration is 50mM). Mix to dissolve. Alternatively, to limit the degree of disulfide reduction, add a 500-fold molar excess of 2-mercaptoethylamine over the concentration of antibody present. 

For reduction of the cystamine disulfides, add 20 pi of 1.0 M DTT and incubate at room temperature for 15 minutes. This will release 2-mercaptoethylamine from the cystamine modification site and create the free sulfhydryl on the 5 terminus of the oligonucleotide. 

The unique characteristics of DTT and DTE are mainly reflected in their ability to form intramolecular ring structures upon oxidation. Disulfide reductants such as 2-mercaptoethanol, 2-mercaptoethylamine, glutathione, thioglycolate, and 2,3-dimercaptopropanol cleave disulfide bonds in a two-step reaction that involves the II formation of a mixed disulfide (Fig. 66). In the second stage of the reducing process, the... 

Reduction of the cystamine-labeled oligo using a disulfide reducing agent releases 2-mercaptoethylamine and creates a thiol group (Fig. 399). DNA probes labeled in this manner have been successfully conjugated with SPDP-activated alkaline phosphatase (Chapter 16, Section 1.5, and Section 2.4), maleimide-activated horseradish peroxidase (section 3.8.1), NHS-LC-biotin (Chapter 8, Section 3.1, and Section 2.3), and the fluorescent tag AMCA-HPDP (Chapter 8, Section 1.3, and Section 2.5). 

Reduce the antibody starting material. Add 2-mercaptoethylamine HC1 (2-MEA, cysteamine HC1) to the antibody solution to a final concentration of 6-7 mg/mL (approx 53-62 mM) while stirring. Dithiothreitol (DTT) at a final concentration of 50 mM also can be used for reduction. 

Numerous reducing agents have been applied for the cleavage of disulfides in peptides. Reduction with sodium in liquid ammonia, with cysteine, 2-mer-captoethanol, 2-mercaptoacetic acid, 2-mercaptoethylamine (cysteamine), borohydrides and trialkylphosphines were used both for preparative and for... 

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