Tris(2-carboxyethyl)phosphine (TCEP) is a widely used reducing agent for the reduction of disulfide bonds on proteins. Some red fluorophores, including Cy5 and Alexa 647, but also the red fluorophores of the Monolith protein labeling kits (MO-L001, MO-L004, MO-L008, MO-L011, MO-L014 and MO-L018) are strongly quenched by high concentrations of TCEP (Fig. 1). The quenching is caused by a reversible addition of TCEP to the dye with a dissociation constant in the mM range. To avoid interference in Monolith binding assays, TCEP should therefore only be used at sub-mM concentrations. If higher concentrations of reducing agent are needed, DTT, β-mercaptoethanol or GSH (glutathione) are recommended.

DTT, β-mercaptoethanol and GSH, however, can interfere with the NHS- and maleimide-labeling reaction and therefore should be avoided. A strategy for circumventing this problem is to label the protein in a buffer with TCEP and then to perform the purification step with column B using a buffer containing DTT.

Figure 1: The K_D for the reversible interaction of TCEP with the RED dye from the Monolith 2nd Generation protein labeling kits is ~10-15 mM. Quenching of capillary fluorescence begins at TCEP concentrations >1 mM.

Apart from the loss in fluorescence intensity and the need for higher target concentrations in the assay to obtain a sufficient fluorescence signal, the reversible fluorescence quenching of TCEP is also strongly temperature-dependent. As a consequence, the IR-laser induced temperature increase in the Monolith shifts the equilibrium of the TCEP-dye interaction towards higher concentrations of TCEP, which can result in a partial recovery of the fluorescence and unusual-looking upward-going MST traces (Fig. 2). This effect on the MST response can therefore disturb the evaluation of target-ligand interactions with MST and lead to issues with reproducibility.

Figure 2: The reversible fluorescence quenching of TCEP is strongly temperature-dependent. The IR-laser induced temperature increase disturbs the equilibrium of the TCEP-dye interaction and the fluorescence recovers, resulting in upward-going MST traces.