Concanavalin a sepharose

Ultrapure guanidinium chloride (GdnCl) and ammonium sulfate were purchased from Calbiochem. Concanavalin A sepharose, HiTrap Q HP column, Superdex 75 pg column and gel filtration molecular mass standards were purchased from GE Healthcare Biosciences Ltd. Biogel P-100 was purchased from BioRad. A protein molecular mass marker set was purchased from Fermentas.

Upon collection, conditioned media (15 mL) from transfected COS-7 cells were pH adjusted with 100 mM Bis-Tris, pH 6.5, and concentrated 15-fold using 30,000 MW cutoff filters (Centricon; Millipore, Billerica, MA) to a final volume of 1 mL. Approximately 1 mL conditioned medium was incubated with 50 µL Concanavalin A-Sepharose (50% slurry; GE Healthcare, Piscataway, NJ) pre-equilibrated with Lysis Buffer (150 mM NaCl, 0.1% Triton X-100, 25 mM Bis-Tris, pH 6.5). The mixture was incubated with rocking for at least 1 hour at 4°C. Beads were collected by centrifugation and washed thrice with Lysis Buffer. Half the beads were used for enzyme activity assays and the other half for SDS-PAGE/immunoblotting as described previously [26] (link).

Native H11 was isolated from adult H. contortus using concanavalin A-sepharose (GE Healthcare), as previously described (8 (link)). Briefly, 15 g of the worms were homogenized in ice-cold phosphate-buffered saline (PBS, pH 7.4) for 25 min using a glass homogenizer. The homogenate was centrifuged (12,000 g for 25 min) and the pellet was extracted four times with 1% (v/v) Thesit in PBS, and filtered (0.45 µM). H11 was isolated using concanavalin A-sepharose columns. The column-bound H11 was washed 3 times (20 mM Tris-HCl), followed by elution using a buffer containing 200 mM of methyl-D-mannopyranoside and methyl-D-glucopyranoside. The resultant solution was enriched and filtered (0.22 µM). This final filtrate was designated the native H11 antigen. Protein concentration was estimated using a bicinchoninic acid assay (BCA) kit (Beyotime Biotechnology), and quality was assessed by SDS-PAGE analysis and Coomassie blue staining.