The gels were stained using Coomassie Brilliant Blue G-250 (GE Healthcare, Uppsala, Sweden). The protein spots were detected, quantified, and matched using ImageMaster 2D Platinum 7.0 analysis software (GE Healthcare, Uppsala, Sweden). Differences in the expression levels between paired samples were analysed, and protein spots showing at least 1.5-fold changes in intensity and anova p < 0.05 were picked for trypsin digestion and MALDI-TOF MS analysis.
Coomassie brilliant blue g 250
Manufactured by GE Healthcare
Sourced in Sweden, United States
Coomassie Brilliant Blue G-250 is a laboratory reagent used for the detection and quantification of proteins. It binds to proteins and produces a blue color that can be measured using a spectrophotometer. This dye-binding assay is a widely used method for protein determination in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Imagemaster 2d platinum 7.0 analysis software, by GE Healthcare (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Enhanced chemoluminescence, by GE Healthcare (1 mentions)
Mini protean 3 electrophoresis cell, by Bio-Rad (1 mentions)
Hybond c extra nitrocellulose membrane, by GE Healthcare (1 mentions)
4 protocols using coomassie brilliant blue g 250
1
Quantitative Proteomic Analysis
2
RBD Protein Characterization by SDS-PAGE and Western Blot
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Purified RBD produced in P. pastoris or in HEK-293T cells was boiled in sample buffer (4% SDS, 20% glycerol, 120 mM Tris, pH 6.8, 0.002% bromophenol blue, 200 mM 2-mercaptoethanol) and separated in 12% SDS-PAGE. Proteins were either stained with Coomassie brilliant blue G-250 or transferred to nitrocellulose membranes (GE Healthcare). The membranes were blocked with 5% milk in 0.05% Tween TBS at room temperature for 1 h and then incubated at 4 °C overnight with a specific polyclonal serum produced by immunization of mice with RBD produced in HEK-293 T cells. Horseradish peroxidase (HRP)-conjugated anti-mouse were incubated for 1 h at room temperature and visualized by enhanced chemiluminescence (ECL, Thermo Scientific).
3
SDS-PAGE Equilibration and Electrophoresis
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Prior to 2D SDS-PAGE, each IPG strip was washed in equilibration buffer 1 (375 mM Tris–HCl pH 8.8, 6 M urea, 2% SDS, 2% DTT) for 15 min followed by equilibration buffer 2 (375 mM Tris–HCl pH 8.8, 6 M urea, 2% SDS, 2.5% iodoacetamide) for 15 min. Each IPG strip plus a SDS-PAGE Molecular Weight Standard (Invitrogen) was loaded on a homogeneous 12% polyacrylamide gel and sealed with 1% agarose. Electrophoresis was performed at 15°C with an initial voltage of 110 V for 30 min, followed by 220 V until the tracking dye reached the gel bottom. All gels were stained with Coomassie brilliant blue G-250 according to the manufacturer’s instructions (GE Healthcare). Each 2D IEF/SDS-PAGE experiment was repeated three times.
4
SDS-PAGE and Western Blot Analysis
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We performed sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions (8% and 12% SDS-PAGE), according to the method of Laemmli [16 (link)]. We used a Mini-Protean 3 electrophoresis cell for the separation of proteins (Bio-Rad Laboratories, Richmond, CA, USA). Proteins were electrophoresed at 120 V, and gels stained with 0.25% Coomassie Brilliant Blue G-250 (GE Healthcare, Waukesha, WI, USA). Pre-stained standards (Fermentas, Burlington, Ontario, CA) were used for estimating the molecular weight of proteins in each sample. Following electrophoresis, proteins were transferred to Hybond-C Extra nitrocellulose membranes (GE Healthcare). We detected β-1,3-glucanosyltransferase and 1,3-β-D-glucan synthase by incubating membranes with the relevant specific murine antibodies (1 mg/mL) overnight. Membranes were then incubated for 1 h with horseradish peroxidase-conjugated anti-mouse IgG (diluted 1:1000 in blocking buffer). The reaction was revealed using the peroxidase substrate ECL (Enhanced Chemo-Luminescence, GE Healthcare).
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