The RED-Tris-NTA dye (MO-L008) and the RED-Tris-NTA second generation dye (MO-L018, MO-L118) are designed to specifically bind the hexa-histidine (6xHis) expression tag of recombinant proteins with high affinity. To establish a binding assay for MST or spectral shift one must first determine the affinity of the dye to the protein. This is done in a simple binding experiment where the RED-Tris-NTA dye serves as target and the unlabeled 6xHis tagged protein as ligand.

Occasionally other polyhistidine tags are used in protein purification, with ten residues (deca-histidine tag) being the most common form. When performing a binding assay of the RED-Tris-NTA dye to the 10xHis tagged protein, it can occur that the initial fluorescence shows a protein concentration dependent biphasic behavior, or simply a drop in fluorescence at intermediate protein concentration as depicted in figure 1.

This is because one dye molecule occupies only 6 histidine residues. At high protein concentration each dye molecule is bound to one protein, resulting in the fluorescence signal of bound dye. At very low protein concentration the majority of dye molecules are unbound, giving rise to the dye’s signal in its unbound state. However, at intermediate protein concentrations some binding of the dye to the other four histidine residues occurs, which can cause quenching.

Figure 1: Illustration of the effect giving rise to a drop in initial fluorescence when measuring the affinity of the RED-Tris-NTA dye to a 10xHis tagged protein. Quenching of capillary fluorescence occurs at intermediate protein concentrations, where the dye is in a modest excess.

This observation has no implications for the further experimental workflow, as the affinity of the dye for the second binding event is in the micromolar region. At standard assay conditions the His-tagged protein is used in 2:1 excess, which favors the one dye per protein state.

Moreover, standard assays are conducted with nanomolar dye and protein concentrations. At those concentrations, even with slight dye excess the number of 10xHis-tagged proteins with two dyes attached is only very small compared to the proteins with one dye. This is due to the difference in affinities between the two “binding sites”, where the affinity for the first dye is usually in the low nanomolar regime.