MA104/NSP5-BioID2 cells (1.5 × 108) were infected with SA11 (MOI, 5), treated at 6 hpi with 100 µM biotin, and lysed 24 h post-transfection in TNN buffer [Tris–HCl 100 mM (pH 8), NaCl 250 mM, and NP-40 0.5% with cOmplete protease inhibitor cocktail (Roche)]. The pull-down of biotinylated proteins was performed using 100 µL StrAv Mag Sepharose (GE Healthcare) for each condition and incubated in a rotation wheel for 3 h at 4°C. For mass spectrometry analysis, the washed biotin pull-downs were digested directly with trypsin (200 ng) in 20 µL of 20 mM triethyl ammonium bicarbonate pH 8.5 for 16 h at room temperature. The supernatant was removed, the beads were washed once with 50 µL of 0.1% formic acid, and the two fractions were pooled and concentrated using STAGE tips (90 (link)). The samples were resuspended in 10 µL of 0.1% formic acid and analyzed by LC-MS/MS using a NanoEASY LC (Thermo) coupled with an amaZon ETD ion trap (Bruker Daltonics). The resulting spectra were searched against the human and rotavirus proteomes using the GPM (91 (link)). Results were filtered to remove all results with an e-value > 0.05. Statistical analysis and plots were performed using R (4.1.3). The figure was finalized in Adobe Illustrator 2022.
Strav mag sepharose
Manufactured by GE Healthcare
StrAv Mag Sepharose is a magnetic bead-based affinity chromatography matrix designed for the purification of streptavidin-tagged proteins. The product consists of agarose beads with covalently coupled streptavidin, which has a high affinity for biotin and biotin-labeled molecules. The magnetic properties of the beads facilitate efficient separation and washing of the captured target proteins.
Automatically generated - may contain errors
2 protocols using strav mag sepharose
1
Rotavirus Host Interactome Profiling
2
Immunoprecipitation and Mass Spectrometry Analysis
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HEK293T cells were transfected with scFv-2A-P* encoding plasmid and lysed 24 h post-transfection in TNN buffer [Tris–HCl 100 mM (pH 8), NaCl 250 mM, NP-40 0.5%], and scFv reporter was immunoprecipitated using mAb anti-SV5. For BioID encoding plasmids, transfection was performed in the presence of biotin and lysed 24 h post-transfection in TNN buffer. Pull-down of biotinylated proteins was performed using StrAv Mag Sepharose™ (GE Healthcare). For mass spectrometry analysis, samples were digested with 200 ng trypsin diluted in 20 mM triethyl ammonium bicarbonate (pH 8.5) for 12 h at room temperature. The supernatants were harvested, and the beads were washed once with digestion buffer. The washes and the supernatants were pooled and purified using STAGE tips. The purified digested samples were subjected to LC–MS/MS analysis using a picofrit nano-bore columns. The sample was loaded in 0.1% formic acid, and the column was developed with a discontinuous gradient and sprayed directly into an Amazon ETD ion trap (Bruker Daltonics). A cycle of one MS scan followed by five MS/MS scans was performed throughout the run. Data were extracted from the runs using Data Analysis (Bruker Daltonics) and analyzed using the X!tandem search engine.
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