Isoelectric focusing was performed using an IPGphor focusing unit (GE Healthcare, Uppsala, Sweden). Samples were loaded on 7 cm long pH 4–7 Immobiline DryStrips (GE Healthcare, Uppsala, Sweden). Strips were rehydrated for at least 24 h with 125 μl of the diluted protein sample containing 125 μg of proteins, 2.5 μl of bromophenol blue solution 0.1%, 2 μl of IPG buffer pH 4–7 (GE Healthcare, Uppsala, Sweden) and 2.5 μl of 1 M dithiothreitol solution in Protein Extraction Buffer-V. Subsequently, pH 4–7 strips were focused at 20°C with a limited current of 50 μA/strip using the following conditions: gradient 0→150 V for 1 h, 150 V for 1 h, gradient 150→300 V for 1 h, 300 V for 2 h, gradient 300→1200 V for 3 h, 1200 V for 1 h, gradient 1200→3500 V for 5 h, and 3500 V for 5.5 h. Strips with pH 6–11 were focused at 20°C with a limited current of 50 μA/strip using the following conditions: gradient 0→150 V for 2 h, 150 V for 1 h, gradient 150→300 V for 1 h, 300 V for 2 h, gradient 300→1200 V for 3 h, 1200 V for 1 h, gradient 1200→3500 V for 5 h, and 3500 V for 1.5 h.
Ipg buffer ph 4 7
Manufactured by GE Healthcare
Sourced in United Kingdom
IPG buffer pH 4–7 is a solution used in isoelectric focusing, a technique in protein separation and analysis. It provides a pH gradient ranging from 4 to 7, which helps separate proteins based on their isoelectric points. The buffer maintains a consistent pH environment during the electrophoresis process.
Automatically generated - may contain errors
Lab products found in correlation
Immobiline drystrip, by GE Healthcare (1 mentions)
Ipgphor focusing unit, by GE Healthcare (1 mentions)
Porcine trypsin, by Promega (1 mentions)
Nuclease mix, by GE Healthcare (1 mentions)
Destreak reagent, by GE Healthcare (1 mentions)
Bluesafe, by NZYTech (1 mentions)
Nzycolour protein marker 2, by NZYTech (1 mentions)
Phosstop, by Roche (1 mentions)
Pepstatin, by Roche (1 mentions)
Complete edta free, by Roche (1 mentions)
Anti phospho serine antibody, by Thermo Fisher Scientific (1 mentions)
Anti acetylated lysine, by Cell Signaling Technology (1 mentions)
Anti peroxiredoxin 2, by Abcam (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Enzo Life Sciences (1 mentions)
Anti phospho threonine, by Cell Signaling Technology (1 mentions)
Mg132, by Merck Group (1 mentions)
Protein g sepharose, by GE Healthcare (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
5 protocols using ipg buffer ph 4 7
1
Isoelectric Focusing of Protein Samples
2
Phosphoproteomic Analysis of Maize Leaves
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All reagents used in this study are analytical or HPLC/MS grade. PhosStop, cOmplete EDTA-free, and pepstatin were acquired from Roche Applied Science (Germany). Nuclease Mix, Destreak Reagent, IPG Buffer pH 4–7, and 7 cm Immobiline® Drystrips pH 4–7 were obtained from GE Healthcare (UK). The phosphoprotein stain Pro-Q® Diamond (PQD) and the PeppermintStick™ phosphoprotein molecular weight markers were purchased from Life Technologies (CA, USA). The whole proteome Coomassie Brilliant Blue stain, BlueSafe (CBB) and the protein molecular weight markers NZYColour Protein Marker II were acquired from NZYTech (Portugal). Porcine trypsin was acquired from Promega Corporation (WI, USA). Seeds from Zea mays inbred line B73 used in this study were amplified in our greenhouse over the years. Original seeds were kindly provided by Dr. Christoph Peterhansel (University of Hannover, Germany).
3
Antibody Reagents for Peroxiredoxin Analysis
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Anti-peroxiredoxin-1, anti-peroxiredoxin-2, anti-peroxiredoxin-3, and anti-peroxiredoxin-SO3 were obtained from Abcam (Cambridge, MA, USA). Anti-peroxiredoxin-6 and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Anti-phosphothreonine and anti-acetylated lysine were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-phosphoserine antibody was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Enzo Life Sciences (Farmingdale, NY, USA). Protein G-Sepharose and IPG buffer pH 4–7 were obtained from GE Healthcare Life Sciences (Pittsburgh, PA, USA). Hydrogen peroxide, MG132, and cycloheximide were purchased from Sigma Aldrich (St. Louis, MO, USA). Lactacystin was obtained from AG Scientific, Inc. (San Diego, CA, USA).
4
Extracellular Protein Isolation and Analysis
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Extracellular proteins were recovered by centrifuging the bacterial cultures (3,500×g) for 15 min at 4°C. The protein concentrations of the S. scabiei culture supernatants were determined according to Bradford (7 (link)) using bovine serum albumin as a standard. In proteomic assays, supernatants were concentrated to a final volume of 500 μL using Amicon Ultra-15 Centrifugal Filters-3K, and the proteins were resuspended in 5 volumes of 100% pre-chilled acetone and kept for 3 h at −20°C. Proteins were recovered by centrifugation (14,000×g, 20 min, 4°C) and the protein pellets were air-dried and resuspended in 80 μL of a buffer composed of 8 M urea, 2% (w/v) CHAPS, 2% (v/v) IPG buffer pH 4–7 (GE Healthcare, Buckinghamshire, UK), 18.15 mM DTT, and 0.002% bromophenol blue stock solution in 50 mM Tris-base. A final centrifugation (14,000×g) was carried out for 5 min at 4°C to remove insoluble material and the protein solutions were immediately subjected to a proteomic analysis.
5
Two-Dimensional Gel Electrophoresis of B. pertussis Lysate
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After the sample preparation, 50 μg B. pertussis B1917 lysate was incubated for 30 min at RT after adding 10 μl 250 mM DTT, 0.62 μl IPG buffer pH 3–10 non-linear (NL), or IPG buffer pH 4-7 (GE Healthcare, Netherlands), and DeStreak Rehydration Solution (GE Healthcare, Netherlands) to a final volume of 115 μl. Immobiline DryStrips pH 3–10 NL, 7 cm, or pH 4–7, 7 cm (GE Healthcare, Netherlands) were rehydrated with sample in an IPGbox (GE Healthcare, Netherlands). Isoelectric focusing (IEF) was performed in the Ettan IPGphor three IEF system (GE Healthcare, Netherlands) with the following program: 0.5 h gradient to 300 V, 1 h gradient to 1,000 V, 1 h 2,000 V, 1 h 3,000 V, 1 h 4,000 V, and 1 h 5,000 V. After IEF strips were equilibrated 15 min in 3 mL equilibration buffer containing 75 mM Tris-HCl pH 8.8, 6 M Urea (Sigma Aldrich, Merck, Germany), 30% Glycerol, 2% SDS, bromophenol blue and 65 mM DTT, followed by a 15 min equilibration with equilibration buffer containing 54 mM iodoacetamide (Sigma Aldrich, Germany) instead of 65 mM DTT. Strip was placed on a 4–12% NuPAGE bis-tris Zoom gel (Thermo Fisher, Invitrogen, Netherlands) and sealed with Agarose sealing buffer (BioRad, Netherlands). Proteins were separated in MES running buffer in a Xcell surelock minicell electrophoresis system for 50 min at 200 V. Gels were stained with Coomassie or used for Western blot.
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