An amount of 10 μg B. pertussis B1917 lysate (see sample preparation) was incubated with 4 × reducing sample buffer (250 mM Tris (Thermo Fisher, Merck, Netherlands), 8% SDS (Sigma Aldrich, Germany), 400 mM DTT (Sigma Aldrich, Germany), 40% Glycerol (Sigma Aldrich, Germany), and 0.04% bromophenol blue (Sigma Aldrich, Germany) 10 min at 100°C. Sample was loaded on 10% NuPAGE bis tris 1.0 mm precast gel (Thermo Fisher, Invitrogen, Netherlands) and proteins were separated with MES running buffer (Thermo Fisher, Invitrogen, Netherlands), 200 Volt (V) for 45 min in Xcell surelock minicell system (Thermo Fisher, Invitrogen, Netherlands). Gel was either stained with Coomassie (Imperial protein stain, Thermo Fisher, Netherlands) or used for Western blot.
Coomassie imperial protein stain
Manufactured by Thermo Fisher Scientific
Sourced in Netherlands, Belgium
Coomassie (Imperial) protein stain is a lab equipment product designed for the detection and quantification of proteins in polyacrylamide gels. It provides a simple, reliable, and sensitive method for visualizing proteins after electrophoresis.
Automatically generated - may contain errors
Lab products found in correlation
Mes running buffer, by Thermo Fisher Scientific (2 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Glycerol, by Merck Group (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Xcell surelock mini cell system, by Thermo Fisher Scientific (1 mentions)
Bromophenol blue, by Merck Group (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Silverquest, by Thermo Fisher Scientific (1 mentions)
Immun blot pvdf membrane, by Bio-Rad (1 mentions)
Bolt 8 bis tris plus gel, by Thermo Fisher Scientific (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Fusion solo s imaging system, by Vilber (1 mentions)
Xcell surelock mini cell electrophoresis system, by Thermo Fisher Scientific (1 mentions)
Fim2 3, by Sanofi (1 mentions)
Agarose sealing buffer, by Bio-Rad (1 mentions)
Nupage novex bis tris zoom gel, by Thermo Fisher Scientific (1 mentions)
4 protocols using coomassie imperial protein stain
1
Denaturing SDS-PAGE of B. pertussis Lysate
2
Extraction and Analysis of LPS from Neonatal Stool
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On the basis of availability of sufficient extracted LPS material, 0.5 µg of extracted LPS from the stool samples of two VD neonates (C007 and C111) collected on day 3 after birth, were prepared with Laemmli sample buffer (Bio-Rad, Belgium), heated for 5 min at 95 °C and separated on 12 % Bis-Tris precast gel (Bio-Rad, Belgium) at 200 V for 45 min. As positive controls, 0.5 µg, 1 µg and 10 µg of commercially available LPS (Escherichia coli O55:B5, gel-filtration chromatography; Sigma-Aldrich, Belgium) and 10 µg of E. coli protein extract were used. A precast gel was loaded with the LPS samples and stained with Coomassie (Imperial protein stain, ThermoFisher, Belgium) to check for protein contaminations. Silver staining of the gel was performed using a corresponding kit (SilverQuest, ThermoFisher, Belgium) according to the manufacturer’s instructions.
3
Characterizing Free FXIII-B Subunit by SDS-PAGE and Western Blot
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In a first step, we analyzed the beads with SDS‐PAGE and western blotting. The beads were resuspended in Laemmli buffer (Bio‐Rad Laboratories) and boiled, and the supernatant was separated by electrophoresis on Bolt 8% Bis‐Tris Plus Gels (Invitrogen) with 1X Bolt™ MES‐SDS Running Buffer (Invitrogen). Gels were stained with Coomassie Imperial Protein Stain (Thermo Fisher Scientific). For Western blotting, the proteins were transferred onto Immun‐Blot® PVDF Membrane (Bio‐Rad) with 1X Bolt Transfer Buffer (Invitrogen). The membranes were incubated with mouse monoclonal anti–free FXIII‐B IgG antibody, followed by a horseradish peroxidase–conjugated goat anti‐mouse secondary antibody, and developed with WesternBright Quantum HRP chemiluminescent substrate (Advansta, San Jose, CA, USA). We visualized the membranes with a Fusion Solo S imaging system (Vilber, Eberhardzell, Germany).
4
SDS-PAGE and 2DE analysis of B. pertussis
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For SDS-PAGE, 10 μg of B. pertussis (B1917) lysate, 1 μg of Prn P.69 (in house prepared 36 ), Ptx (Kaketsuken, Japan), FHA (Kaketsuken, Japan), or Fim2/3 (Sanofi) was incubated (10 min, 100 °C) with 3.3 μL of reducing sample buffer (250 mM Tris, 8% SDS, 400 mM DTT, 40% glycerol, 0.04% bromophenol blue) and loaded on a 10% NuPAGE bis tris 1.0 mm precast gel (Invitrogen). For 2DE, the equilibrated strip was placed on a 4-12% NuPAGE Novex bis-tris ZOOM gel (Invitrogen) and sealed with agarose sealing buffer (BioRad). Proteins were separated (SDS-PAGE, 45 min, 200 V) (2DE, 50 min, 200 V) with MES running buffer (Invitrogen) in a Xcell surelock minicell electrophoresis system (Invitrogen). Gels were washed in water and either stained with Coomassie (Imperial protein stain, Thermo Scientific), used for Western blot ,or scanned using an Odyssey infrared imager (Westburg).
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