Biotinylated vva

For VVA ELISA, whole-cell lysates were prepared using RIPA-B lysis buffer. RIPA-B was exchanged with PBS using centrifugal filter units (Merck Millipore) which was repeated 4 times. 40 μg total proteins were measured using BCA protein assay kit and coated onto the ELISA microplate (Greiner bio-one, Neuburg, Germany) for 16 h at 4 °C. The total bound proteins were washed with PBS containing 0.05% Tween 20 and treated with blocking solution (Vector Labs, Burlingame, CA) for 2 h at 4 °C. Each well was washed three times and added with 10 μg ml⁻¹ biotinylated VVA (Vector Labs) for 16 h at 4 °C. Target proteins bound to biotinylated VVA were stained using Vectastain ABC kit (Vector Labs). Absorbance was measured using a microplate spectrophotometer (Berthold technologies, Wildbad, Germany) at 405 nm.