2 μg of mycobacterial protein lysates per lane were separated on NuPAGE Novex 4–12% Bis-Tris ZOOM Gels (Invitrogen) using NuPAGE MES SDS Running Buffer (Invitrogen) under reducing conditions. After electrophoresis proteins were transferred onto nitrocellulose membranes using an iBlot gel transfer device (Invitrogen). Membranes were blocked with blocking buffer (5% non-fat dry milk in PBS) ON at 4°C. Membranes were then incubated in blocking buffer containing anti-MUL_3720 IgG (mouse mAbs JD3.2, JD3.4 or rabbit polyclonal IgG SZ3398) or mouse mAb DD3.7 (specific for a conserved mycobacterial protein) serving as loading control for 1 h at RT. After washing, membranes were incubated with secondary goat anti-mouse IgG (γ-chain specific) (HRP, SouthernBiotech) or goat anti-rabbit IgG (Fc fragment specific) (HRP, Milan) for 45 min at RT. After washing, bands were visualized by chemiluminescence using the ECL Western Blotting substrate (Pierce).
Nupage novex 4 12 bis tris zoom gels
Manufactured by Thermo Fisher Scientific
NuPAGE Novex 4–12% Bis-Tris ZOOM Gels are pre-cast polyacrylamide gels designed for protein electrophoresis. They feature a Bis-Tris buffer system and a 4–12% gradient separation range.
Automatically generated - may contain errors
Lab products found in correlation
Iblot gel transfer device, by Thermo Fisher Scientific (2 mentions)
Nupage mes sds running buffer, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated goat anti human igg γ chain specific, by Southern Biotech (1 mentions)
Nap 5 column, by GE Healthcare (1 mentions)
Ettan ipgphor, by GE Healthcare (1 mentions)
Mark12, by Thermo Fisher Scientific (1 mentions)
3 protocols using nupage novex 4 12 bis tris zoom gels
1
Quantitative Detection of MUL_3720 Protein
2
Western Blot Analysis of Recombinant Proteins
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Western Blot analysis was performed as described [11 (link)]. Shortly, 15 μg of recombinant M. ulcerans 18 kDa shsp or P. falciparum AMA-1 were separated on NuPAGE Novex 4–12% Bis-Tris ZOOM Gels with 1.0 mm IPG well (Invitrogen) under reducing conditions. After electrophoresis, proteins were transferred onto nitrocellulose membranes using an iBlot Gel Transfer Device (Invitrogen). Membranes were blocked with 5% non-fat dry milk in PBS containing 0.1% Tween 20 and cut into strips. Strips were then incubated with human blood sera (1:1000 dilution), washed with 0.3 M PBS containing 1% Tween 20 and thereafter incubated with horseradish peroxidase conjugated goat anti-human IgG (γ-chain specific, Southern Biotech). After a second washing step, bands were visualized by chemiluminescence using ECL Western Blotting substrate (Pierce).
3
Fungal Secretome Proteomic Analysis
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Fungal culture supernatants were desalted on a NAP-5 column (GE Healthcare) and 50 μg protein was precipitated by adding four volumes of ice-cold acetone and dissolved in 125 μL rehydration buffer (7 M urea, 2 M thiourea, 2% (w/v) CHAPS, 200 mM destreak reagent (bis (2-hydroxyethyl) disulfide; GE Healthcare), 0.5% (v/v) pharmalytes pH range 3-10 (GE Healthcare), trace of bromophenol blue). The samples were applied to 7 cm pH 3-10 IPG strips (GE Healthcare) for isoelectric focusing (IEF) (Ettan™ IPGphor; GE Healthcare) after rehydration (12 h at 50 mA/strip at 20°C), performed to reach a total of 20 kVh (1 h at 150 V, 1 h at 300 V, 1 h at 1000 V, gradient to 8000 V, held at 8000 V until a total of 20 kVh). The strips were equilibrated (2 15 min) in 5 mL equilibration buffer (6 M urea, 30% (v/v) glycerol, 50 mM Tris HCl, pH 8.8, 2% (w/v) SDS, 0.01% (w/v) bromophenol blue) supplemented with 1% (w/v) dithiothreitol and 2.5% (w/v) iodoacetamide in the first and second equilibration steps, respectively. The strip and molecular weight marker (Mark 12, Invitrogen) were placed on NuPAGE Novex 4-12% Bis-Tris Zoom gels (Invitrogen) and run according the manufacturer's instructions. Gels were stained with colloidal Coomassie Blue (G-250). 2D-gel electrophoresis was performed in duplicate (2 biological replicates, Supplementary Figure S1).
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