For the gel‐based proteomic profiling of chemically cross‐linked protein species in wild type versus dystrophic mdx‐4cv skeletal muscle, analytical grade reagents and materials were purchased from GE Healthcare (Little Chalfont, Buckinghamshire, UK), Sigma Chemical Company (Dorset, UK), Bio‐Rad Laboratories (Hemel‐Hempstead, Hertfordshire, UK) and National Diagnostics (Atlanta, GA, USA). Protease inhibitor cocktails were obtained from Roche Diagnostics (Mannheim, Germany). The chemical cross‐linker BS³ and C18 spin columns were supplied by Thermo Fisher Scientific (Dublin, Ireland). Proteolytic digestion was carried out with sequencing grade modified trypsin from Promega (Madison, WI, USA). Biobasic C18 Picofrit columns were from Dionex (Sunnyvale, CA, USA).
Biobasic c18 picofrit column
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
The Biobasic C18 Picofrit column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of analytes. It features a silica-based C18 stationary phase and a picofrit design, which provides improved chromatographic performance and reduced sample dispersion.
Automatically generated - may contain errors
Lab products found in correlation
Sequencing grade modified trypsin, by Promega (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
C18 spin column, by Thermo Fisher Scientific (2 mentions)
Q exactive, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Memcode, by Thermo Fisher Scientific (1 mentions)
Polyvinylpyrrolidone 40, by Merck Group (1 mentions)
Ponceau s red staining solution, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Dionex rslcnano, by Thermo Fisher Scientific (1 mentions)
Rc dc protein assay, by Bio-Rad (1 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (1 mentions)
5 protocols using biobasic c18 picofrit column
1
Proteomic Profiling of Dystrophic Muscle
2
Gel Electrophoresis Reagents Protocol
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Analytical grade chemicals and materials for gel electrophoresis were obtained from Amersham Biosciences/GE Healthcare (Little Chalfont, Buckinghamshire, UK), National Diagnostics (Atlanta, GA, USA) and BioRad Laboratories (Hemel-Hempstead, Hertfordshire, UK). Protease inhibitor cocktails were purchased from Roche Diagnostics (Mannheim, Germany). Nitrocellulose membranes were from Millipore (Bedford, MA, USA). The reversible membrane stain Memcode was purchased from Thermo Fisher Scientific (Waltham, MA, USA) and sequencing grade modified trypsin was obtained from Promega (Madison, WI, USA). Liquid chromatography-mass spectrometry Chromasolv water was purchased from Fluka (Milwaukee, WI, USA). Biobasic C18 Picofrit columns were from Dionex (Sunnyvale, CA, USA) and C18 spin columns were obtained from Thermo Fisher Scientific (Dublin, Ireland). N-acetylglucosamine agarose, Ponceau S-Red staining solution, polyvinylpyrrolidone-40 and formic acid, as well as all other analytical grade chemicals used in this study, were purchased from Sigma Chemical Company (Dorset, UK).
3
Proteomic Analysis of Multiple Myeloma Patient Samples
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BM-MNC CD138+ cells from 23 MM patient samples were lysed in RIPA buffer (Cell Signaling Technology, Danvers, MA, USA), and proteins digested. An amount of 500 ng of each digested whole-cell protein lysate was loaded onto a Q Exactive (Thermo Fisher Scientific, Hemel Hempstead, UK) high-resolution accurate mass spectrometer connected to a Dionex Ultimate 3000 (RSLCnano) chromatography system (Thermo Fisher Scientific, Hemel Hempstead, UK). Peptides were separated using a 2% to 40% gradient of acetonitrile on a Biobasic C18 Picofrit column (Thermo Fisher Scientific, Hemel Hempstead, UK) (100 mm length, 75 mm internal diameter) for over 65 min at a flow rate of 250 nL/min. Data were acquired with the mass spectrometer (MS) operating in automatic data-dependent switching mode. A full MS scan at 140,000 resolution and a range of 300–1700 m/z was followed by an MS/MS scan at 17,500 resolution and a range of 200–2000 m/z, selecting the 10 most intense ions prior to MS/MS.
4
High-Resolution Mass Spectrometry Analysis
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500 ng of each digested sample was loaded onto a Q-Exactive (ThermoFisher Scientific, Hemel Hempstead, UK) high-resolution accurate mass spectrometer connected to a Dionex Ultimate 3000 (RSLCnano) chromatography system (ThermoFisher Scientific, Hemel Hempstead, UK). Peptides were separated using a 2% to 40% gradient of acetonitrile on a Biobasic C18 Picofrit column (ThermoFisher Scientific, Hemel Hempstead, UK) (100 mm length, 75 mm ID) over 65 min at a flow rate of 250 nl/min. Data was acquired with the mass spectrometer operating in automatic data dependent switching mode. A full MS scan at 140,000 resolution and a range of 300–1700 m/z was followed by an MS/MS scan, resolution 17,500 and a range of 200–2000 m/z, selecting the ten most intense ions prior to MS/MS37 (link).
5
Quantitative Proteomic Workflow for Cell Lysates
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Cell pellets were lysed in a buffer containing 8 M urea/50 mM NH4HCO3/0.1% ProteaseMax. The protein amount was estimated using an RC/DC protein assay from Bio-Rad27 (link). BSA was used as a standard. After dithiothreitol reduction and iodoacetic acid-mediated alkylation, a double digestion was performed using Lys-C (for 4 hours at 37 °C) and Trypsin (overnight at 37 °C) on 5 µg of protein. Digested samples were desalted prior to analysis using C18 spin columns (Thermo Scientific, UK). 500 ng of digested protein was analysed from each digest using a Q-Exactive mass spectrometer coupled to a Dionex RSLCnano (Thermo Scientific, Waltham, MA, USA). Peptides were separated using a 2% to 40% gradient of acetonitrile (A: 0.1% FA, B: 80% acetonitrile, 0.1% FA) on a Biobasic C18 Picofrit column (ThermoFisher Scientific, Hemel Hempstead, UK) (100 mm length, 75 mm ID) over 65 min at a flow rate of 250 nl/min. Data was acquired with the mass spectrometer operating in automatic data dependent switching mode. A full MS scan at 140,000 resolution and a range of 300–1700 m/z was followed by an MS/MS scan, resolution 17,500 and a range of 200–2000 m/z, selecting the 15 most intense ions prior to MS/MS (Top15 method)28 (link).
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