α1 2 fucosidase

NGLs or glycolipids (2 nmol) were dissolved in 70 μl of 50 mm sodium citrate buffer, pH 6.0, containing 0.1 mg/ml bovine serum albumin (BSA) and 0.5 mg/ml sodium cholate. After a brief sonication, 200 units of α1–2 fucosidase (New England Biolabs) were added, and the mixture was incubated at 37 °C for 48 h. The reaction mixture was purified by a C18 cartridge, and the products were eluted with CHCl₃/MeOH/H₂O, 30:70:30.

Anti-TRA-1-60 (clone TRA-1-60, mouse IgM) was obtained from R&D Systems Inc. (Minneapolis, MN, USA). Horseradish peroxidase (HRP)-conjugated rabbit anti-mouse Ig was obtained from Agilent Technology (Santa Clara, CA, USA). Polyclonal goat anti-podocalyxin IgG and HRP-conjugated rabbit anti-goat IgG secondary antibodies were obtained from R&D Systems. Anti-human iPSC/ESC, R-10G (mouse IgG1), and R-17F (mouse IgG1) antibodies were prepared as described previously [6 (link),8 (link)].
Peptide N-glycanase (PNGase F; recombinant protein from Escherichia coli) was obtained from Roche Diagnostics GmbH (Mannheim, Germany), neuraminidase (Arthrobacter ureafaciens) from Nacalai Tesque (Kyoto, Japan), α1-3/4 fucosidase from TaKaRa Bio, Inc. (Shiga, Japan), and α1-2 fucosidase from New England Biolabs (Ipswich, MA, USA). Chondroitinase ABC (Proteus vulgaris), heparinase mixture (heparinase, heparitinase I, and heparitinase II), keratanase (Pseudomonas sp.), keratanase II (Bacillus sp.), and endo-β-galactosidase (Escherichia freundii) were obtained from Seikagaku Biobusiness (Tokyo, Japan).

For on-array glycosidase digestion, the array was hydrated with HEPES-buffered saline (HBS, 5 mm HEPES, pH 7.4, 150 mm NaCl, 5 mm CaCl₂) for 2 min and blocked with 3% bovine serum albumin (BSA, Sigma) in HBS buffer for 1 h before addition of α1,2-fucosidase (New England Biolabs, Hitchin, UK, P0724L Lot 0111505) in 50 mm sodium citrate buffer, supplemented with 100 mm NaCl and 1 μg/ml BSA (pH 6.0). The enzyme was evaluated at 8–20 unit/μl and incubation was at 37 °C for 12–44 h. The binding analyses were performed after treatment with the enzyme at 8–20 unit/μl. The array was then washed 3 times with HBS and once with water and dried at ambient.