Tgs running buffer

Infected Vero cells from single-cycle infections were harvested in 1X NuPage LDS sample buffer (Thermofisher) diluted in PBS containing protease inhibitor (Roche). Cell lysates were homogenized using a QIAshredder spin column (Qiagen) and protein concentrations were determined by BCA assay (Pierce BCA Protein Assay kit). Thirty μg of proteins was denatured in a final composition of 1X NuPage LDS sample buffer (ThermoFisher) and 1X NuPAGE Sample Reducing Agent (Invitrogen) by heating at 90°C for 10 min before being subjected to electrophoresis on NuPAGE 4–15% Mini-PROTEAN TGX Gels (Bio-Rad) with 1X TGS Running Buffer (Bio-Rad). Odyssey Protein Molecular Weight Marker (Li-Cor) was run in parallel. Proteins were transferred to PVDF membranes using the iBlot 2 Gel Transfer Device (ThermoFisher) and stained with a primary mouse anti-RSV F (abcam, ab43812), a mouse anti-RSV P (abcam, ab94965) or a mouse anti-RSV G (abcam, 94966) antibody. The rabbit anti-Tubulin (abcam, ab52866n) antibody was used as a loading control. The secondary antibodies used were goat anti-rabbit IgG IRDye 680RD (at 1:15,000, Li-Cor, 926–68071), and goat anti-mouse IgG IRDye 800CW (1:10,000, Li-Cor, 926–32210). Membranes were scanned using Odyssey software, version 3.0 (Li-Cor).

Two-dimensional gels were run using 250 µg of whole cell lysate with 0.2% pH 3–10 carrier ampholytes (Bio-Rad). Isoelectric focusing was performed using 11 cm pH 4–7 IPG strips (Bio-Rad) and 11 cm pH 6–11 immobiline drystrips (GE Healthcare). Focusing was carried out using a Protean IEF system (Bio-Rad) at a constant 20°C and 50 µA current limit per strip with a three-step programme: slow ramp to 4000 V for 4 h, linear ramp to 10 000 V for 4 h, then 10 000 V until 120 kVh was reached. Following IEF, the strips were equilibrated with 5 ml equilibration solution (2% SDS, 6 M urea, 250 mM Tris–HCl pH 8.5, 0.0025% (w/v) bromophenol blue) for 20 min before the second-dimension SDS–PAGE. The second-dimension gels were run using precast Bio-Rad TGX midi gels with TGS running buffer (Bio-Rad). Reference gels were stained with Coomassie blue G250 overnight and destained with 1% acetic acid to remove background. All visible spots (180 from the pH 4–7 gel and 160 from the pH 6–11 gel) were manually excised from the gel and subjected to in-gel trypsin digestion, before analysis by LC–MS/MS.