Synergy plate reader

ELISA plates (Immulon^® 4HBX, Thermo Scientific) were coated with 2 μg/mL protein (goat anti-human lambda chain antibody or recombinantly expressed HA glycoprotein. Recombinant HAs included were derived from A/PR/8/34 (H1N1), A/California/04/09 (H1N1), A/Indiana/10/11 (H3N2), A/chicken/Netherlands/14015531/14 (H5N8) and A/chicken/Italy/13474/99 (H7N1) [12 (link), 13 (link)]) and incubated at 4°C overnight. Next day, plates were incubated with 3% milk in phosphate-buffered saline (PBS, pH7.4)) with 0.1% Tween 20 (PBST) for 2 hours at room temperature (RT), shaking. After the plates were washed, cell culture supernatant was applied and incubated for 1 hour at RT, shaking. Goat anti-human Fab antibody conjugated to horse-radish peroxidase (Sigma) was used as a secondary antibody in 1:3000 dilution. O-phenylenediamine dihydrochloride (OPD) (Sigma) was provided as a substrate for visualization and plates were measured using a Synergy plate reader at an optical density of 490 nm.

Integrity and purity of purified YqhD protein were checked on a native tris-bis gel (4–16%, Thermo Fisher Scientific) using 1X Native Page Running Buffer (Invitrogen). The gel was stained with Coomassie Plus Protein Assay Reagent (Thermo Fisher Scientific) for 1 h, distained overnight in water, and photographed with Bio-Rad gel doc EZ imager. The enzymatic activity of purified YqhD was determined using the method adapted from Pérez et al. (2008) (link) and measured using a Synergy plate reader at 37°C, in 200 μL of 50 mM phosphate buffer (pH 7.0) supplemented with 0.6 mM NADPH (Sigma-Aldrich), 10 μg of purified YqhD, and 0–4 mM of glutaraldehyde. The K_M was calculated by OriginPro (OriginLab) using inbuilt Michaelis–Menten function.