Pro q emerald 300 glycoprotein gel and blot stain kit

Beef was purchased at a local butcher's store, shock‐frozen with liquid nitrogen, reduced to small pieces with a mortar and stirred in PBS containing protease inhibitors (Roche Diagnostics GmbH, Rotkreuz, Switzerland) overnight at 4°C. Thereafter, the extract was centrifuged at 10 000 g for 30 min and the supernatant was filtered through filter paper (Macherey‐Nagel, Düren, Germany), lyophilized and stored at −20°C. The protein concentration was determined by bicinchoninic acid assay (Bio‐Rad Laboratories, Richmond, CA, USA). The extract (20 μg) was separated by 12% SDS‐PAGE under nonreducing conditions and stained with Coomassie brilliant blue (Bio‐Rad Laboratories). Detection of glycosylation was performed with the Pro‐Q^® Emerald 300 Glycoprotein Gel and Blot Stain Kit (Thermo Fisher Scientific) according to the manufacturers’ protocol. For immunoblot experiments, the separated extract was transferred to a nitrocellulose membrane. After blocking, sera were incubated overnight at 4°C. Bound IgE was detected with ¹²⁵I‐labelled anti‐human IgE antibody (Demeditec Diagnostics, Kiel‐Wellsee, Germany) and visualized by autoradiography. Buffer and sera of nonallergic donors served as negative controls.

P. clara 1C4 was cultured overnight in the presence of Tunicamycin (Sigma-Aldrich; final concentration, 10 µg ml⁻¹), 2-fluro-l-fucose (Cayman Chemical; final concentration, 250 µM) or DMSO control. Cultured bacteria were then pelleted, washed once with PBS and lysed with 1% SDS solution (in 50 mM Tris-HCl buffer supplemented with 5 mM EDTA). SDS–PAGE was conducted using the Novex NuPAGE SDS–PAGE Gel system (Thermo Fisher Scientific). Glycan-containing proteins were stained with the Pro-Q Emerald 300 Glycoprotein Gel and Blot Stain Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. The protein contents of the whole-cell lysates were stained using the Colloidal Blue Staining kit (Thermo Fisher Scientific). Supernatant proteins were first condensed using Amicon Ultra Centrifugal Filters (10 kDa NMWL) and then stained using the Colloidal Blue Staining kit (Thermo Fisher Scientific).

Purified pili and molecular weight markers (Precision Plus Protein™ All Blue Prestained Protein Standards, Biorad, Hercules, USA; CandyCane glycoprotein molecular weight standards, Thermo Fisher, Waltham, USA) were separated by SDS-PAGE (Mini Protean TGX 4–20%, Biorad). Protein was stained using InstantBlue™ Protein Stain (Expedeon Ltd, Cambridge, UK) or Pro-Q™ Emerald 300 Glycoprotein Gel and Blot Stain Kit (Thermo Fisher). For the lectin analysis, after SDS-PAGE proteins were transferred to PVDF membranes (Trans-Blot Turbo System, Biorad). Blots were blocked and incubated with biotinylated lectins (10 μg/ml; Vector Laboratories, Burlingame, USA) according to the manufacturer’s instructions, followed by incubation with IRDye 800CW Streptavidin (LI-COR, Lincoln, USA) and imaging using an Odyssey Fc (LI-COR).