Cells were lysed for 15 min at 4°C in lysis buffer (20 mM Tris HCl, pH 7.5, 150 mM NaCl, 0.5% NP-40, 50 mM NaF, and 1 mM sodium orthovanadate, supplemented with complete protease inhibitor cocktail; Roche Diagnostic). Lysates were centrifuged at 10,000 g for 10 min at 4°C. The postnuclear supernatants were kept and an equal amount of proteins (BCA dosage kit; Pierce) was analyzed by SDS-PAGE. Immunoprecipitation was performed with HeLa cell lysates incubated with 4 µg rabbit anti–HA antibody for 1 h, followed by a 3-h incubation with Protein G beads. Proteins were transferred onto polyvinylidene difluoride membrane (Millipore) and incubated in blocking solution PBS/0.1% Tween-20 supplemented with 5% milk for 2 h. Blots were rinsed with PBS/0.1% Tween-20 and antibodies were incubated in the blocking solution. Detection was performed using ECL substrate (GE Healthcare). For mass spectrometry analysis, digestion was performed on beads and samples were desalted (ZipTip C18) and then analyzed with an nanoESI-Orbitrap on the LTQ-Orbitrap Fusion with nano-LC Proxeon 1000 (Thermo Fisher Scientific; Mass Spectrometry Facility, Institut Jacques Monod, Paris, France).
Nano lc proxeon 1000
Manufactured by Thermo Fisher Scientific
Sourced in France
The Nano-LC Proxeon 1000 is a liquid chromatography system designed for nano-scale separations. It features a low-flow pumping system capable of delivering mobile phases at nano-flow rates, enabling the analysis of small sample volumes. The system is optimized for coupling with mass spectrometry for sensitive biomolecular detection and identification.
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Lab products found in correlation
2 protocols using nano lc proxeon 1000
1
Immunoprecipitation and Mass Spectrometry
2
Proteomic Analysis of Antibody Effects
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Proteins on beads from 3 experimental conditions (control, mouse antibody, rabbit antibody) were digested in biological triplicates overnight at 37 °C by sequencing grade trypsin (12.5 µg/ml; Promega Madison, WI, USA) in 20 µl of 25 mM NH4HCO3. Peptides were desalted using ZipTip µ-C18 Pipette Tips (Thermo Fisher Scientific). Peptide mixtures were analyzed by a Q-Exactive Plus coupled to a Nano-LC Proxeon 1000 (both from Thermo Fisher Scientific). Peptides were separated by chromatography as performed previously31 (link). In brief, the following parameters were used: Acclaim PepMap100 C18 pre-column (2 cm, 75 μm i.d., 3 μm, 100 Å), Pepmap-RSLC Proxeon C18 column (50 cm, 75 μm i.d., 2 μm, 100 Å), 300 nl/min flow rate, a 98 min gradient from 95% solvent A (water, 0.1% formic acid) to 35% solvent B (100% acetonitrile, 0.1% formic acid). Peptides were analyzed in the Orbitrap cell, at a resolution of 70,000, with a mass range of m/z 375–1500. Fragments were obtained by higher-energy collisional dissociation (HCD) activation with a collisional energy of 28%. MS/MS data were acquired in the Orbitrap cell in a Top20 mode, at a resolution of 17,500.
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