Western Blot Analysis of Influenza HA Proteins

Ten ng of recombinant H4 HA and H11 HA (negative control) prepared in PBS were mixed with equal volume of 2x Laemmli loading buffer (Bio-Rad) supplemented with 2% beta-mercaptoethanol (BME) and heated at 100°C for 15–20 min. Samples were then run on sodium dodecyl sulphate (SDS) polyacrylamide gels (5–20% gradient; Bio-Rad) and later transferred onto nitrocellulose membranes. The remaining procedure was directly adapted from a published protocol [55 (link)]. Membranes were blocked with 3% non-fat milk in TPBS and then stained with 30 μg/mL of each antibody prepared in 1% non-fat milk in TPBS. Membranes were then washed three times with TPBS and stained with anti-mouse IgG (whole molecule)–alkaline phosphatase (AP) antibody produced in goat (Sigma-Aldrich). Reactivity was visualized by treatment of membranes with development solution prepared from AP conjugate substrate kit (Bio-Rad).