Goat anti mouse irdye conjugated secondary antibody

Preparations were run on 12% Novex NuPAGE Bis-Tris SDS-PAGE gels (Life Technologies) before being transferred to Hybond-C Extra nitrocellulose membrane (GE Healthcare). Membranes were probed with mouse antiserum against 6xHisTag (1:5000, Abcam), or mouse antiserum against CD2831^{20 (link)}, followed by goat anti-mouse IRDye conjugated secondary antibody (1:2000, LI-COR Biotechnology). Blots were visualised with an Odyssey near-infrared imager (LI-COR Biotechnology).

Cultured cells were lysed in Laemmli sample buffer containing 0.1 M DTT and heated for 5 min at 95 °C, followed by loading onto a 10% sodium dodecyl sulfate polyacrylamide gel and separation by electrophoresis for 90 min at 120 V, after which the proteins were electrophoretically transferred onto a polyvinylidene difluoride membrane (Invitrogen) for 1.5 h with an electric current of 250 mA. Subsequently, the membrane was blocked with a mixture of 2.5 ml of blocking buffer (Odyssey, USA) and 2.5 ml of phosphate-buffered saline containing 0.05% Tween 20. This was followed by overnight incubation with anti-HEV capsid protein primary antibodies (1:1000) at 4 °C. The membrane was then washed three times, followed by incubation for 1 h with goat anti-mouse IRDye-conjugated secondary antibody (Li-COR Biosciences, Lincoln, USA) (1:5000). After washing three times, protein bands were detected using an Odyssey 3.0 Infrared Imaging System.