Nupage mes sds running buffer

Huh-7.5 cells infected with intergenotyic core viruses at 10 TCID50/cell were lyzed at 72 h, 96 h, or 120 h post-infection in NuPAGE LDS sample buffer (Life Technologies) and HEK293 cells transfected with core expressing DNAs were lyzed at 48 h post-transfection in a CHAPS buffer (Tris 50 mM pH7.5, NaCl 140 mM, EDTA 5 mM, Glycerol 5%, CHAPS 1%), respectively, containing 0.71 M 2-mercaptoethanol, protease inhibitor (Roche) and phosphatase inhibitor (Pierce) cocktails. Protein extracts were incubated for 10 min at 95 °C (NuPAGE LDS extracts) or at 65 °C (CHAPS extracts), loaded onto NuPAGE 4–12% Bis-Tris gels (Life Technologies), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in NuPAGE MES SDS running buffer (Life Technologies), and transferred onto polyvinylidene difluoride (PVDF) or nitrocellulose membranes (GE Healthcare Life Sciences). Membranes were saturated in PBS containing 0.1% Tween-20 (PBS-T) and 5% dry skimmed milk at room temperature for 1 h and incubated overnight at 4 °C with primary antibodies diluted in PBS-T containing 1% dry skimmed milk. Primary antibodies used are listed in Supplementary Table 3. Following incubation with appropriate secondary antibodies conjugated to DyLight 800 or 680 (Thermo Scientific), proteins were visualized by using the Odyssey CLx imaging system (Li-Cor Biosciences, Lincoln, Nebraska. USA).

An equal volume/amount of protein samples was prepared in 4X sodium dodecyl sulfate (SDS) loading buffer containing 100 mM dithiothreitol (DTT) (Astral, NSW, Australia). Samples were then subjected to denaturation by heating at 95 °C for 2 min. The denatured samples were run on a NuPAGE^® 4–12% Bis-Tris precast gel (Life Technologies, Mulgrave, VIC, Australia) at 150 V for 1 h in the presence of NuPAGE^® MES SDS Running Buffer (Life Technologies, Mulgrave, VIC, Australia). Proteins were then transferred to mini nitrocellulose membranes using iBlot dry blotting system (Invitrogen™, Mulgrave, VIC, Australia) for 7 min at 20 V. The membrane was then blocked with skim milk 5% (w/v) in Tris-buffered saline with 0.05% (v/v) Tween 20 (TTBS) for 45 min. The membrane was washed with TTBS three times (10 min each) and probed with required primary antibodies at 1:1000 dilution overnight at 4 °C. The blot was then washed three times over 30 min with TTBS and then subsequently with appropriate IRDye (LI-COR^®, Lincoln, NE, USA) conjugated secondary antibodies (1:10,000) for 1 h at room temperature. The probed blot was then washed three times with TTBS (10 min each). The protein bands were visualized using ODYSSEY Clx (LI-COR^®, Lincoln, NE, USA).

In the analyses of proteins produced in the 50 mL flask cultivations, SDS-PAGE was run using the standard Laemmli method as described in [42 ].
Nu-PAGE Bis-Tris Mini Gels (4–12% gradient) and NuPAGE LDS sample buffer with NuPAGE reducing agent (Life Technologies, Carlsbad, CA, USA) were used for the analysis of samples derived from the 500 mL cultures and bioreactor productions. Furthermore, the accompanying NuPAGE MES SDS running buffer (Life Technologies) was used.

Cell lysates were prepared in RIPA buffer, and equal amounts of protein were loaded into NuPage 4–12% Bis-Tris Midi gels (Invitrogen) submerged in NuPage MES SDS Running Buffer (Life Technologies). The proteins were transferred onto a nitrocellulose membrane using the SureLock™ Tandem Midi Blot Module (Life Technologies) and NuPage Transfer Buffer (Life Technologies) containing 10% methanol. The membrane was blocked with 5% powdered milk in tris-buffered saline for 1 hour at room temperature. Membranes were incubated with either a mouse monoclonal anti-alpha smooth muscle actin (Abcam ab7817, 0.341ug/mL) or a rabbit monoclonal anti-vimentin antibody (Abcam ab92547, 1:5000 dilution) with a mouse monoclonal anti-GAPDH loading control (Arigo biolaboratories ARG10112, 1:5000 dilution) overnight at 4°C. Incubation with HRP secondary antibodies (Arigo biolaboratories ARG65350, 1:5000 dilution or enQuire BioReagents QAB10303, 1:15,000 dilution) was performed for 1 hour at room temperature. Signal was produced using SuperSignal^TM West Pico PLUS Chemiluminescent Substrate (Thermo Scientific) and the blots were imaged on a LI-COR Fc Imager (Odyssey).

Bacterial samples required for Western blotting were grown aerobically overnight in M9 minimal medium at 37°C. The following day cultures were subcultured and grown in M9 minimal medium at 37°C to approximately mid‐logarithmic growth phase (OD_600nm ~0.6) then harvested by centrifugation, and cell pellets were re‐suspended in 50 mM Tris–HCl (pH 8.0). Protein extracts were prepared by sonication on ice with an MSE Soniprep 150 (Sanyo, UK) for four pulses of 30 s with a 30‐s pause between each pulse. A Bradford assay was carried out to quantify the protein concentration, and 10 μg of protein was run on 4–12 % NuPAGE^® Bis‐Tris mini gels with NuPAGE^® MES SDS running buffer (Life Technologies, UK). Protein was transferred to nitrocellulose transfer membranes (Whatman, UK), and analyzed by Western blotting using AcrB antibody at a 1 : 1,000 dilution. Blots were developed using anti‐rabbit IgG horseradish peroxidase‐linked antibody (Sigma, UK) at a 1 : 25,000 dilution and analyzed using the ECL detection system (GE Healthcare UK).