Western blot analysis

The amount of glycoprotein produced in periplasmic and membrane fractions was analysed by Western blot. Samples were resuspended in SDS-PAGE loading buffer (ThermoFisher), supplemented with 75 mM dithiothreitol, and boiled for 10 min. Equal amounts of sample were separated by SDS PAGE (Biorad) and Western blotted onto PVDF 0.2 µM pore size membrane using a Turbo Transblot apparatus (Biorad). The membrane was blocked with 5% (w/v) milk powder in PBS (30 min, 50 rpm shaking, room temperature). Membranes were incubated overnight (50 rpm, 4 °C) with mouse monoclonal anti-His antibody (1:3000 dilution in PBS 5% milk, Pierce) and rabbit polyclonal anti-Cj glycan (CjNgp) primary antibody(1:500 dilution in PBS 5% milk) as appropriate. Membranes were washed three times with PBS, followed by incubation with anti-mouse and anti-rabbit secondary antibody (1:30,000 dilution in PBS 5%, Li-Cor) (50 rpm, in the dark at room temperature, 30 min), and then washed three times with PBS. The protein bands were visualised with a Li-Cor Odyssey scanner. Proteins were quantified based on densitometry analysis using Li-Cor Image Studio 5.0. Pre-determined purified protein from each sample (scFv13R4, RNase A, or NGRP) was used as a standard for quantification (5 ng to 100 ng). All data were produced in biological triplicate.