Protein-Glycan Binding Assay Protocol

Biotinylated protein was incubated overnight with Alexa 488-labeled streptavidin (Invitrogen) at a ratio of ∼2 mol of CRD to 1 mol of streptavidin subunit. The mixture was applied to a 1-ml column of mannose-Sepharose, which was washed with loading buffer, and the complex was eluted with 0.5-ml aliquots of elution buffer. The protein was tested against version 5.1 of the glycan array of the Consortium for Functional Glycomics using the standard protocol.
Competition binding assays were performed as previously described for mincle (22 (link)). ¹²⁵I-Man-BSA and ¹²⁵I-IgG reporter ligands were prepared by radioiodination (23 (link)) of Man₃₁-BSA (E-Y Laboratories) and human IgG (Sigma).