Coronavirus nucleocapsid proteins assemble constitutively in high molecular oligomers

Cong, Yingying; Kriegenburg, Franziska; de Haan, Cornelis A. M.; Reggiori, Fulvio

For the pull-down experiments, GSH-Sepharose bound GST fusion protein were incubated with 200 µl of bacterial extract or 200 µl of LR7 cell extracts on a rotatory wheel for 2 h at 4 °C, subsequently washed at 4 °C three times in PBS supplemented with 5 mM DTT, 10% glycerol, 1% Triton X-100 and one time in PBS buffer. Proteins bound to the Sepharose beads were eluted in 20 µl of sample buffer by boiling and subjected to SDS-PAGE, blotted onto PVDF membranes and visualized by either membrane staining with Ponceau Red or western blot analysis using anti-6xHis antibody (HIS H8, Thermo, Waltham, MA) or anti-N protein monoclonal antibodies 11 . Bound primary antibodies were detected using the Alexa680-conjugated goat polyclonal anti-mouse IgG antibody (Life Technologies) and signals visualized with an Odyssey system (LI-COR, Lincoln, NE).