90 μg of trichloroacetic acid (TCA) precipitated M. ulcerans (NM20/02) protein lysate was resuspended in rehydration buffer (8 M urea, 2% 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 0.5% (v/v) ZOOM Carrier Ampholytes (Invitrogen), 0.002% bromophenol blue and 0.4% dithioerythritol (DTE)). The mix was incubated with a 3–10 pH gradient strip (ZOOM Strip; Invitrogen) over night (ON) at room temperature (RT). First-dimension isoelectric focusing (IEF) was performed on a ZOOM IPG runner (Invitrogen) using a step voltage protocol (175 V for 15 min, 175–2000 V for 45 min, 2000 V for 2 h). After IEF, the strips were incubated for 15 min with equilibration buffer (6 M urea, 50 mM Tris pH 8.8, 30% glycerol, 2% SDS, 30 mM DTE) followed by a 15 min incubation period with alkylating solution (6 M urea, 50 mM Tris (pH 8.8), 30% glycerol, 2% SDS, 0.23 M iodacetamide). Second-dimension gel electrophoresis was performed at 200 V for 35 min using a 10% NuPAGE Novex Bis-Tris ZOOM Gel (Invitrogen). The gel was stained with Coomassie blue (Invitrogen).
Nupage novex bis tris zoom gel
Manufactured by Thermo Fisher Scientific
The NuPAGE Novex Bis-Tris ZOOM Gel is a pre-cast polyacrylamide gel used for protein separation and analysis in electrophoresis. The gel is formulated with a Bis-Tris buffer system and provides high-resolution separation of proteins.
Automatically generated - may contain errors
Lab products found in correlation
Coomassie blue, by Thermo Fisher Scientific (1 mentions)
Zoom strip, by Thermo Fisher Scientific (1 mentions)
Coomassie imperial protein stain, by Thermo Fisher Scientific (1 mentions)
Fim2 3, by Sanofi (1 mentions)
Agarose sealing buffer, by Bio-Rad (1 mentions)
Mes running buffer, by Thermo Fisher Scientific (1 mentions)
Xcell surelock mini cell electrophoresis system, by Thermo Fisher Scientific (1 mentions)
2 protocols using nupage novex bis tris zoom gel
1
Proteomic Analysis of M. ulcerans
2
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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