Mini protean tgx polyacrylamide gradient gels

Harvested aliquots of purified RSV were boiled for 5 min with Laemmli sample buffer and 2.5% 2-mercaptoethanol, then run on precast Mini-PROTEAN TGX polyacrylamide gradient gels (Biorad, Mississauga, ON, Canada) at 150 V for 1 h in tris-glycine-sodium dodecyl sulfate running buffer. Coomassie gels were stained with 3 g/L Brilliant Blue R-250, destained with 45% methanol, 45% water, and 10% acetic acid (v/v/v), then imaged. For the Western blot analysis, proteins were transferred onto a nitrocellulose membrane at 100 V for 1 h at 4 °C in tris-glycine transfer buffer including 10% methanol. The membranes were washed with 0.1% Tween (TBS-T), blocked with 5% bovine serum albumin (BSA) in TBS-T, and probed with primary then secondary-HRP antibodies in TBS-T containing 1% BSA. Protein bands were detected by ECL 2 substrate (Pierce PI80196), and imaged using a GE ImageQuant LAS 4000 (GE Healthcare Life Sciences).

To collect protein lysates, cells were chilled, washed with ice-cold PBS and then lysed with either MOSLB buffer or using the NE-PER (Thermo Fisher, 89842) fractionation kit. Collected protein lysates were heated to 100°C for 10 min with 1×Laemmli sample buffer supplemented with 2.5 % 2-mercaptoethanol as a reducing agent. Samples were loaded on precast Mini-PROTEAN TGX polyacrylamide gradient gels (Biorad) and ran at 150 V for 1 h in Tris-glycine-SDS running buffer. Separated proteins were wet-transferred onto a nitrocellulose membrane at 100 V for 1 h at 4 °C in Tris-glycine transfer buffer supplemented with 10 % methanol. Membranes were washed with Tris-buffered saline containing 0.1 % Tween (TBS-T), blocked with 5 % BSA in TBS-T for 1 h at room temperature. Membranes were probed with primary antibodies, followed by secondary antibodies conjugated to HRP, in TBS-T containing 1 % BSA. Chemifluorescence was generated by adding ECL2 substrate (Pierce pi80196) to blots for 5 min at room temperature, then removed for blots to be imaged. Blots were imaged with a BioRad ChemiDoc MP, detecting HRP using the Cy2 filter and markers using the Cy5 filter. Adobe Photoshop or ImageJ was used to adjust image brightness and contrast.