Reagents disulfide-reducing

Cleland (1964) showed that DTT and DTE are superior reagents in reducing disulfide bonds in proteins (see previous discussion, this section). DTT and DTE have low oxidation-reduction potential and are capable of reducing protein disulfides at concentrations far below that required with 2-mercaptoethanol. However, even these reagents have to be used in approximately 20-fold molar excess in order to get close to 100 percent reduction of a protein. 

Dissolve the antibody to be biotinylated in 50 mM sodium bicarbonate, pH 8.5, at a concentration of 10 mg/ml. Other buffers and pH conditions between pH 7 and 9 can be used as long as no amine-containing buffers like Tris are present. Avoid also the presence of disulfide reducing agents that can cleave the disulfide group of the biotinylation reagent. 

The protocol for using isobaric tags differs from that described previously for the ICAT or ECAT type reagents. In the following method, the proteins are denatured and the disulfides reduced and then alkylated to block them permanently. This eliminates disulfide re-association and also prevents the isobaric tags from forming thioester modification with cysteine thiols. Next, the proteins are digested with trypsin and then modified with an isobaric tag. Each sample is labeled with a different isobaric compound so that the samples can be differentiated upon MS/MS analysis. 

Thiols can be detected on insoluble supports by treatment with the symmetric disulfides of 4-mercaptonitrobenzene or 5-mercapto-2-nitrobenzoic acid ( Ellman s reagent [69,70]). These reagents are reduced by thiols to the corresponding thiophe-nolates, which are intensely colored. 

Sodium disulfide (or polysulflde) solution, Na S. For use in the preparation of thio-salicylic acid, the reagent is prepared by heating and stirring a mixture of 1.1 moles of Na2S-9H20, 290 ml. of water, and 1.1 moles of powdered sulfur until a brownish red solution results. A solution of 40 g. of sodium hydroxide in 100 ml. of water is added, and the solution is cooled in a salt-ice bath and stirred at 0-5° during addition of a solution of diazotized anthranilic acid. This Sandmeyer-type displacement gives a disulfide reducible to thiosalicylic acid. 

---