Computational protocol: TANGO1 and Mia2/cTAGE5 (TALI) cooperate to export bulky pre-chylomicrons/VLDLs from the endoplasmic reticulum

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[…] Peptides were removed from silver-stained gel bands with iodoacetamide precipitation. After digestion with trypsin, peptides were acidified and desalted before LC-MS/MS analysis. 45% of each sample was analyzed using a LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific) coupled to an EasyLC (Thermo Fisher Scientific). All data were acquired with Xcalibur software. Proteome Discoverer software suite (Thermo Fisher Scientific) and the Mascot search engine (Matrix Science) were used for peptide identification and quantification. The data were searched against the human SwissProt database. Resulting data files were filtered for ion score 20. [...] Cells grown on coverslips were fixed with cold methanol for 10 min at −20°C and incubated with blocking reagent (Roche) for 30 min at RT. Primary antibodies were diluted in blocking reagent and incubated overnight at 4°C. Secondary antibodies conjugated with Alexa Fluor 488, 555, 594, or 647 (Invitrogen) were diluted in blocking reagent and incubated for 1 h at RT. Images were taken with a TCS SPE or TCS SP5 confocal microscope (Leica) with a 63× objective. Two-channel colocalization analysis was performed using ImageJ, and the Manders correlation coefficient was calculated using the plugin JaCop (). […]

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