Difficult to Reduce Proteins
Most proteins attain their 3D conformation, driven by the presence of cysteine amino acids, which form specific disulfide Bonds, which twist and turn the proteins to spatial conformations, many of which ultimately will react with their corresponding receptors on the cell’s surface of their receptors, thus initiating a number of biochemical cascades such us phosphorylation events , and direct trigger signals that make the cell produce specific molecules with specific functions . At times the biochemical researcher needs to reduce the protein, for a variety of reasons, weather to carry on protein sequence analysis, folding-refolding studies, protein activation, denaturation, solubilization and for other reasons.
Commonly used reducing reagents are, dithiothreitol (DTT), 2-mercaptoethanol (MCE)and 2-mercaptoethylamine, and Tris(carboxyethyl) phosphine (TCEP).
The researcher can select which reducing agent to use based upon his/her specific needs, for example, low concentrations of 2-Mercaptoethylamine•HCl are used to reduce the disulfide bridges of antibodies; on the other hand 0.5% solutions of BME are used when preparing samples for isolectric focusing applications. A good alternative to BME is DTT that can be used at concentrations ranging from 10-100mM (pH 7-9).
TCEP has the advantage of being across a larger pH range (2-11) and posses a greater redox potential as compared to DTT. In real case situations, one can run into difficult to reduce proteins, whereby one hast to use even harsher reducing conditions like 10 mM DTT and 8M urea, 4hrs ambient or 6M guanidine, 80 mM DTT, 60min at 40C, followed by alkylation.Hence you have to be careful not to carbamylate(do not heat urea)and deamidation can occur at higher temperature and pH
The following is a good reference:
Wu, J., and Watson, J. T. (1997) A novel methodology for assignment of disulfide bond pairings in proteins, Protein Sci. 6, 391-398.