Dengue Virus E Protein Detection

Protein harvested from C6/36 cells that were either infected by DENV2 or not was boiled for 3 min, then separated by electrophoresis on 12% (w/v) sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) in nonreducing conditions. It was subsequently transferred onto an Immobilon-P Transfer Membrane (Millipore, Darmstadt, Germany). After blocking with 5% milk-TBS-0.1% Tween 20 buffer at RT for 1 h, the membrane was incubated with the indicated primary and secondary antibodies at RT for 1 h as the method done previously in this lab [29 (link)]. Specific primary antibodies included the 4G2 monoclonal antibody (a kind gift of Dr. Guey-Chuen Perng, National Cheng Kung University, Taiwan) for the dengue E protein and anti-actin mouse monoclonal antibody clone C4 (Merck Millipore, Burlington, MA, USA). Secondary antibodies were goat anti-rabbit or anti-mouse IgG antibodies, depending on the primary antibody used in the experiment. After the final wash, the membrane was treated with a Western Lightning Chemiluminescence Plus Reagent (PerkinElmer, Waltham, MA, USA), from which signals were detected on a FUJI X-ray film.