Polyvinylidene difluoride membranes immune blot

Cells were suspended in SDS polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer after being washed with phosphate-buffered saline (PBS). Samples were boiled for 5 min (MP-12 samples) or 15 min (ZH501 samples) and subjected to SDS-PAGE. Proteins were electroblotted onto polyvinylidene difluoride membranes (immune blot: Bio Rad). After blocking with skim milk for 1 h at room temperature, the membranes were incubated with the primary antibody overnight at 4°C, followed by incubation with the secondary antibody for 1 h at room temperature. The membrane was washed three times with PBS containing 0.01% Tween 20 between each incubation step. ECL Western Blotting Detection Reagent (GE Healthcare Life Sciences) or ECL plus Western Blotting Substrate (Pierce) was used for detection of blotted proteins. For detection of Gn, NSm, and N proteins, anti-Gn mouse monoclonal (4D4) [23 (link)], anti-NSm [15 (link)] and anti-N [15 (link)] antibodies were used. Anti-NSm antibody was also used for detection of P78, as it detects both P78 and NSm [15 (link)].

Cells were washed with phosphate-buffered saline (PBS) and suspended in SDS polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. Samples were boiled for 3–5 min and subjected to SDS-PAGE. Proteins were electroblotted onto polyvinylidene difluoride membranes (immune blot: Bio Rad). After blocking with skim milk, the membranes were incubated with the primary antibody for 1 h at room temperature and with the secondary antibody for 1 h at room temperature. The proteins on the membrane were detected by using an ECL Western Blotting Detection Reagent (GE Healthcare Life Sciences) or ECL plus Western Blotting Substrate (Pierce).