Selected article for: "Bis Tris Invitrogen nupage gradient gel and collision energy"
Author: Prashali Bansal; Johannes Madlung; Kristina Schaaf; Boris Macek; Fulvia Bono
Title: An interaction network of RNA-binding proteins involved in Drosophila oogenesis
  • Document date: 2020_1_9
    • ID: 2f9nc2to_12
    Snippet: For proteome measurements, eluates were separated on a NuPAGE Bis-Tris precast 4-12% gradient gel (Invitrogen). Samples were run approximately 2 cm into the gel and bands were visualized with a 0.1% Colloidal Coomassie Blue stain (Serva). Proteins were digested in-gel using trypsin. Peptides were desalted . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-.....
    Document: For proteome measurements, eluates were separated on a NuPAGE Bis-Tris precast 4-12% gradient gel (Invitrogen). Samples were run approximately 2 cm into the gel and bands were visualized with a 0.1% Colloidal Coomassie Blue stain (Serva). Proteins were digested in-gel using trypsin. Peptides were desalted . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.01.08.899146 doi: bioRxiv preprint Interactome of RNA Binding Proteins from Drosophila ovaries 8 and purified on C18 StageTips (68) . LC-MS analysis were performed on a nanoLC (Easy-nLC 1200, Thermo Fisher Scientific) coupled to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific) through a nanoelectrospray ion source (Thermo Fisher Scientific), as described previously (69) . In brief, peptides were eluted using a segmented gradient of 10%-50% HPLC solvent B (80% ACN in 0.1% formic acid) at a flow rate of 200 nL/min over 46 min. MS data acquisition was conducted in the positive ion mode. The mass spectrometer was operated in a data-dependent mode, switching automatically between one full scan and subsequent MS/MS scans of the 12 most abundant peaks selected with an isolation window of 1.4 m/z (mass/charge ratio). Full-scan MS spectra were acquired in a mass range from 300 to 1650 m/z at a target value of 3 × 10 6 charges with the maximum injection time of 25 ms and a resolution of 60,000 (defined at m/z 200). The higher-energy collisional dissociation MS/MS spectra were recorded with the maximum injection time of 45 ms at a target value of 1 × 10 5 and a resolution of 30,000 (defined at m/z 200). The normalized collision energy was set to 27%, and the intensity threshold was kept at 1 × 10 5 . The masses of sequenced precursor ions were dynamically excluded from MS/MS fragmentation for 30 s. Ions with single, unassigned, or six and higher charge states were excluded from fragmentation selection.

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