Total carbohydrate colorimetric assay kit

Periodic acid-Schiff staining of purified antibodies was done using SDS-PAGE with a glycoprotein staining kit (Thermo Fisher Scientific Pierce) following the manufacturer’s instructions. Equal protein loading was confirmed by SDS-PAGE followed by immunoblot analysis with HRP-conjugated goat anti-mouse IgA (Bethyl). Chemiluminescence signal was detected using a Luminescent Image Analyzer LAS-4000 (Fuji). The amounts of total carbohydrate on purified IgA clones were determined by using the Total Carbohydrate Colorimetric Assay Kit (BioVision). Lectin microarray was performed as described (Inoue et al., 2013 (link)). Briefly, protein was labeled with Cy3 and then applied to lectin array (LecChip; GP Biosciences). The lectin signals were measured with GlycoStation Reader 1200. Scanned images were analyzed with Array-Pro Analyzer (Media Cybernetics) and GlycoStation Tools (GP Biosciences). Lectin concentration was normalized to the total protein concentration measured with a Micro BCA Protein Assay Kit (Thermo Fisher Scientific Pierce).

Observation and measurements of lipids in cells were performed by Oil Red O staining using a Lipid Assay kit (Cosmo Bio, Tokyo, Japan). Carbohydrates were observed by PAS staining24 (link) and their levels were measured using the phenol-sulfuric acid method with a Total Carbohydrate Colorimetric Assay kit (BioVision, Milpitas, CA). Colorimetry of lipids and carbohydrates was carried out at 540 and 490 nm, respectively, using an InfinitM200 (TECAN, Mannendorf, Switzerland).