Peroxidase conjugated streptavidin

The molecular mass of the neo-glycoproteins was analyzed by SDS-PAGE followed by streptavidin blot. Here, protein amounts of 1 µg for Coomassie staining and 0.5 µg for blotting were applied using 8% gels and constant current of 25 mA. The proteins transferred to the PVDF-membrane were detected by incubation with peroxidase-conjugated streptavidin (Roche, Mannheim, Germany).

The culture supernatant obtained from P388D1 cells stimulated for 48 h and assayed for IL-12 and IL-10 by ELISA. Briefly, ELISA plates were coated overnight at 4 °C with purified anti–IL-10 (2 μg/ml) and IL-12 (2 μg/ml). Plates were washed three times (0.05% Tween 20 in PBS) and blocked for 2 h with blocking buffer (1% BSA). After washing the plates for three times, the plates were incubated overnight with standards or culture supernatants. Following this, plates were washed and incubated with respective biotin-conjugated detection antibodies for 1 h at 25 °C. Washed the plates again and incubated with peroxidase-conjugated streptavidin (Roche Applied Science) for 30 min followed by washing and color development using TMB substrate (BD Pharmingen, San Diego, CA). Reaction was stopped by the addition of 1 N H₂SO₄, and absorbance was measured at 450 nm. The values of standards were plotted and the quantity of the cytokines were estimated in the samples and expressed in pg/ml.

TcdB was biotinylated via EZ-Link^TM Sulfo-NHS-Biotin (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Briefly, TcdB was incubated with Sulfo-NHS-Biotin for 30 min at room temperature (RT). To remove the excess non-reacted biotin, TcdB was rebuffered with a Zeba^TM Spin Desalting Column (Thermo Fisher Scientific, Waltham, MA, USA). The successful biotinylation was verified via Western blotting and detection with peroxidase-conjugated streptavidin (1:2500, F. Hoffmann-La Roche AG, Basel, Switzerland). The biotinylated TcdB showed unimpaired activity in a cytotoxicity assay.