To quantify the binding affinities between DC-SIGN and Gx-psDiMan of varying psDiMan content (0 -100%), Atto-643 labeled DC-SIGN (varying concentrations) was mixed with 1 molar equivalent of Gx-psDiMan in a binding buffer (20 mM HEPES, 100 mM NaCl, 2 mM CaCl2, pH 7.8) containing 1 mg/mL of BSA, and then incubated for 20 min at room temperature. The protein concentration ranged from 0.1, 0.2, 0.5, 1, 2, 4, 8 to 16 nM for x = 5, or 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 to 6.4 nM for x = 13, respectively. Fluorescence emission spectra were recoded over a range of 650-800 nm using a fixed EX of 630 nm. Fluorescence spectra of labeled DC-SIGN at the above concentrations without Gx-psDiMan were also recorded under the identical conditions. The fluorescence spectra from 650 to 800 nm were integrated and used to calculate the quenching efficiency (QE) at each concentration (C) using eq. 1. The obtained QE -C plots were fitted by the Hill's equation (eq. 2) to derive their apparent binding Kds.