The mutant proteins have been shown to retain faithfully the tetrameric structure and glycan-binding properties of full-length lectins. We then labeled the lectins using a maleimide-modified Atto-643 dye with a labeling efficiency of > 80% per monomer (SI, Fig. ). Then, we measured the fluorescence spectra of the labeled lectins alone and their 1:1 molar mixtures with G5-glycans over a range of concentrations and fitted the obtained fluorescence quenching efficiency (QE) -concentration relationships using the Hill's equation to derive their apparent Kds. While this method worked well with the small G5-glycans, due to its relatively low absorption extinction coefficient ( = 1.10 × 10 7 M -1 cm -1 ), unfortunately, it cannot work for the larger GNP-glycans. This is because the GNP's  roughly increases linearly with volume, where the very strong absorption (hence inner filter effect) of the large GNPs (e.g.,  = 1.10 × 10 7 , 2.32 × 10 8 and 2.39 × 10 9 M -1 cm -1 for G5, G13, and G27, respectively) can severely interfere with the fluorescence quenching measurement, especially at high concentrations (e.g. >10 nM).