The addition of a reducing agent to an assay may be important to prevent the oxidation of cysteines in the investigated proteins and thereby the formation of Disulfides. The most commonly used reducing agents are dithiothreitol (DTT), β-mercaptoethanol (β-ME) and tris(2-carboxyethyl)phosphine (TCEP).
It has been shown that strong reducing agents such as DTT and TCEP can generate hydrogen peroxide (H2O2) by a chain reaction of oxidation-reduction (redox) cycling in the presence of redox cycling compounds. This can lead to false results and these agents should therefore be used with caution. Several important classes of protein targets like protein tyrosine Phosphatases, cysteine proteases (cathepsins and caspases), and metalloenzymes are susceptible to H2O2-mediated inactivation.
In addition, TCEP reacts with and quenches fluorescence of many red Fluorophores, and should not be used in these cases. Thus, the use of strong reducing agents is not recommended. Weaker reducing agents such as reduced glutathione (GSH), β-ME and cysteine do not induce the generation of H2O2. We recommend 1-5 mM GSH as a reducing agent.