The soluble extracellular segments of DC-SIGN/R (denoted as DC-SIGN/R hereafter) have been shown to faithfully replicate the tetrameric structures and glycan binding properties of full length DC-SIGN/R, therefore they were employed to investigate the pH dependency of DC-SIGN/R binding interactions with Gx-DiMan. To facilitate the sensitive fluorescence readout, the residue Q274 in DC-SIGN or R287 in DC-SIGNR was mutated to a cysteine residue for site-specific dye labelling. These residues are located at close to, but outside of, the glycan binding pocket on the CRD, hence dye labelling does not affect their glycan binding properties, as confirmed previously. The constructed DC-SIGN Q274C and DC-SIGNR R287C, were expressed in E. coli, purified by affinity column chromatography, and then site-specifically labelled with maleimide-modified Atto-643 as described previously. We have chosen Atto-643 here because of its high fluorescence quantum yield, excellent photo-stability, and strong hydrophilicity (minimising the possibility of forming aggregates and inducing non-specific interactions). Moreover, its fluorescence is stable over a pH range of 2 to 11, making it a robust fluorescence probe under a wide range of biological conditions. Furthermore, its absorption and emission occur in the red region of the spectrum where GNPs have minimal absorption, greatly reducing any possible contribution of quenching from GNP's inner filter effect. The dye labelling efficiency was estimated as ∼82% and ∼90% per monomer for DC-SIGN and DC-SIGNR, respectively (see SI, Fig. ).