Microseal f foil

The mucin mimetic bead library was transferred to PCR plates (twin.tec PCR plates LoBind, Eppendorf). Each glycan structure was placed at 1.4 ×10⁶ beads/well in three wells, washed three times with 100 µl PBS and resuspended in 50 µl PBS. All washes were done on a DynaMag-96 side skirted magnetic beads separator plate (Life Technologies), the PCR plate was shifted back and forth from left to right 10 times to mix the beads. IAV (50 µl) was added to the beads and the plate was sealed with microseal ‘F’ foil (Biorad). The plate was vortex-mixed and incubated at 4°C for 2h on a rocking shaker. Incubation was done at 4°C in order to inhibit enzymatic NA activity, however similar results were obtained by 1h incubation at 37°C. The beads were washed three times with PBS to remove unbound virus. Bead-bound virus was quantified by measuring viral NA activity, as described above with minor changes. The beads were resuspended in 60 µl of 0.1mM 4MU-Neu5Ac in 33 mM MES/CaCl₂/NaCl₂ buffer, and incubated for 1 h at 37°C in dark. Following incubation, 50 µl of the supernatant was transferred to 96-well plate, and 150 µl of 25% ethanol, 0.1M glycine pH 10.7 was added. Signal from 50 µl 0.1mM 4MU-Neu5Ac was subtracted from all of the samples. The amount of released 4MU compound was measured at excitation 365 nm and emission 450 nm in a SpectraMax M3 spectrophotometer.