Binding Specificity of BKPyV VP1s

The binding specificities of the his-tagged recombinant BKPyV VP1s were analyzed in the neoglycolipid (NGL)-based microarray system.³⁴ (link) Two versions of microarrays were used: (1) ganglioside-focused arrays featuring 25 glycolipid and NGL probes (Figure 2A), and (2) broad spectrum screening microarrays of 672 sequence-defined lipid-linked glycan probes, of mammalian and non-mammalian type essentially as previously described.³⁵ (link) The glycan probes included in the screening arrays and their sequences are given in Table S2. Details of the preparation of the glycan probes and the generation of the microarrays are in Supplementary Glycan Microarray Document (Table S3) in accordance with the MIRAGE (Minimum Information Required for A Glycomics Experiment) guidelines for reporting of glycan microarray-based data.³⁶ (link) The microarray analyses were performed essentially as described.³ (link)^,37 (link) In brief, after blocking the slides for 1h with HBS buffer (10 mM HEPES, pH 7.4, 150 mM NaCl) containing 0.33% (w/v) blocker Casein (Pierce), 0.3% (w/v) BSA (Sigma-Aldrich) and 5 mM CaCl₂, the microarrays were overlaid with the VP1 proteins for 90 min as protein-antibody complexes that were prepared by preincubating VP1 with mouse monoclonal anti-polyhistidine and biotinylated anti-mouse IgG antibodies (both from Sigma) at a ratio of 4:2:1 (by weight) and diluted in the blocking solution to provide a final VP1 concentration of 150 μg/mL. Binding was detected with Alexa Fluor-647-labelled streptavidin (Molecular Probes) at 1 μg/mL for 30 min. Unless otherwise specified, all steps were carried out at ambient temperature. Microarray imaging and data analysis are described in the Supplementary MIRAGE document (Table S3).