Nupage gel system

Raw peanut extracts were analysed by 1D PAGE using the Nu-PAGE gel system (Invitrogen, Paisley, UK). LDS sample buffer was added (1:4 sample to buffer ratio (v/v)), and samples (1 mg protein/mL) were heated for 5 min at 70 °C. Samples (10 μL per well) and molecular weight markers (Mark-12, Invitrogen, Paisley, UK) were loaded onto 4–12% Bis-Tris precast gradient gels, and proteins were separated at 200 V for 35 min. Gels were stained using Simply Blue Safe Stain (Invitrogen, Paisley, UK) and visualised using a Typhoon TRIO variable mode imager (GE Healthcare, Buckinghamshire, UK). Densitometry was performed using the ImageQuant Software (GE Healthcare, Buckinghamshire, UK) to determine relative abundance of individual allergens.

Cells and tissues were lysed in RIPA-Buffer supplemented with proteinase inhibitors (complete mini, RAS) [23] (link). 30 µg-protein samples were separated using the NuPAGE Gel system (4-12% Tris-Glycine gel; Invitrogen, Karlsruhe, Germany) and transferred to a nitrocellulose membrane (BioRad, Munich, Germany). Blots were blocked with 5% milk powder in TBS, incubated with mouse LHM2 or rabbit H-300 anti-CSPG4 antibodies overnight at 4°C, followed by the peroxidise-conjugated secondary anti-mouse or anti-rabbit antibodies for 1 hour, and exposed to a chemiluminescence detection kit (all reagents from ECL Amersham; GE Healthcare Europe GmbH, Freiburg, Germany). For equal loading control analysis, the membrane was exposed to stripping buffer (Thermo Scientific, Rockford, IL, USA) for 30 min at 37°C, and then incubated with an antibody against GAPDH (36 kDa) or beta-actin (42 kDa) (SCBT). Commercial SK-MEL-28 melanoma cell lysate (SCBT) and freshly prepared HeLa cells lysate were used as positive controls for CSPG4 detection.

The proteins were separated by gel electrophoresis using the NuPAGE gel system (Invitrogen) according to the manufacturer’s protocol. Protein bands of interest were cut and the proteins therein were digested in-gel as previously described48 (link). Alternatively, the proteins were precipitated with ethanol and the protein pellet was dissolved in 10 μl 1% (m/v) RapiGest SF Surfactant (Waters). The proteins were reduced by addition of 10 μl 25 mM dithiothreitol and incubation at 37 °C for 1 h. Free cysteine residues were alkylated with 10 μl 100 mM iodoacetamide at 37 °C for 1 h. The RapiGest concentration was reduced to 0.1% (m/v) by addition of 25 mM ammonium bicarbonate. The proteins were digested with 0.5 μg Trypsin (Promega) at 37 °C overnight. RapiGest was decomposed by addition of 5% (v/v) trifluoroacetic acid and removed by centrifugation. The peptides were dried in a vacuum centrifuge.

Western blot using SDS-PAGE electrophoresis was performed using NuPAGE gel system (Invitrogen) as described in our previous studies^{74 (link),75 (link)}. Briefly, 30 µg (SIRT1) and 10 µg (cleaved caspase 3) of PFC brain lysates from all the animals were separated on a 4–12% and 10% Bis-Tris gels respectively under reducing conditions followed by transfer and immunodetection. Nonspecific antibody binding was blocked using Superblock (Thermofisher Scientific, Rockford, IL, USA) for 1 h at room temperature. Immunoblotting was carried out with antibodies against SIRT1 (1∶1000, Santa Cruz Biotechnology, Santa Cruz, CA) and cleaved caspase-3 (1: 1000, Cell Signaling,Technology, Danvers, MA) followed by secondary antibody (1∶3,000 HRP conjugated anti rabbit IgG for SIRT1 and 1∶3000 HRP conjugated anti rabbit for cleaved caspase-3 respectively (Thermofisher Scientific, Rockford, IL, USA). Blots were developed with 1∶1 solution of Super Signal West Pico Chemiluminescent Substrate and Luminol/Enhancer (Thermo Fisher Scientific, Rockford, IL, USA).

25 µl of supernatant along with known amounts of purified ovine elafin protein were reduced with 1% dithiothreitol and loaded on 4–12% gradient polyacrylamide gel using the Invitrogen NuPage gel system as recommended by the manufacturer. After running the gel proteins were transferred to Hybond ECL Nitrocellulose membrane (Amersham Pharmacia). Resultant membranes were blocked in 5% skimmed milk powder in TPBS (PBS and 0.1% Tween 20). Membranes were probed overnight at 4°C with Trab-2O monoclonal anti-elafin antibody (HyCult Biotechnology, Uden, the Netherlands) diluted 1 in 500–1000 in TPBS or anti-FLAG monoclonal antibody diluted 1 in 1000 in TPBS. Membranes were then washed in PBS-T before the secondary antibody was applied (Goat anti mouse IgG conjugated to HRP). This was followed by final washing, addition of Western LightningTM Chemiluminescent Reagent Plus (PerkinElmer Life Sciences, Inc.), and exposure to X-omat radiograph-quality film (Kodak).

To prepare total cell extract, N. meningitidis strains were grown overnight on GC agar plates at 37°C in 5% CO₂. Colonies from each strain were collected and used to inoculate GC broth to an initial optical density at 600nm (OD600) of 0.05–0.06. The culture was incubated at 37°C with shaking until an OD600 0.5 was reached, and then centrifuged for 5 minutes at 8000 × g. The supernatant was discarded and the pellet was resuspended in SDS-containing sample loading buffer. Total lysates were boiled for 5 minutes, separated by SDS-PAGE using the NuPAGE Gel System (Invitrogen) and transferred onto nitrocellulose membranes for Western blot analysis. Western blots were performed according to standard procedures. The different NHBA peptides were identified with polyclonal mouse antisera raised against the recombinant NHBA protein, corresponding to MC58 protein (peptide 3) (diluted 1:1000). Anti-PilE mouse polyclonal serum was used to determine the strain piliation status and a monoclonal anti-Opc antibody (B306) was used to evaluate Opc expression. In all cases, an anti-mouse antiserum conjugated to horseradish peroxidase (Dako) was used as the secondary antibody. Bands were visualized with Super Signal West Pico Chemiluminescent Substrate (Pierce) following the manufacturer’s instructions.