SINV-nsP3 mScarelt-tagged immunoprecipitation

1x107 cells HEK293 FLP-IN T-REX cells were infected with SINVnsP3-mScarlet (bait) or SINVwt (negative control) at 1 MOI. After 18 hours, cells were scrapped and spin down at 300 g 4°C for 10 mins. After the centrifugation, supernatants were discarded and cell pellet washed once with 1X PBS, followed by another centrifugation at 300xg 4°C for 10min. After discarding the supernatant, cell pellets were lysed with 200µL ice-cold lysis buffer (10mM Tris-Cl pH 7.5, 150mM NaCl, 0.5mM EDTA, 0.5% IGEPAL, benzonase(1U/mL) and 0.1mM AEBSF serine protease inhibitor) , and incubated on ice for 30min. Then, cell lysates were cleared by centrifugation 17,000xg for 10min at 4°C. The supernatants were transferred to new tubes and diluted 1:1 with the dilution buffer (10mM Tris-Cl pH 7.5 150mM NaCl 0.5mM EDTA 0.05% IGEPAL). Next, cell lysates were incubated with 15µL of RFP-Trap® Magnetic Particles M-270 (pre-washed once with the dilution buffer) for 1 hour at 4 °C with mild rotation. After the incubation, the magnetic beads were washed five times with wash buffer1 (10mM Tris/Cl pH 7.5, 150mM NaCl, 0.5mM EDTA and 0.25% IGEPAL), followed by two washes with the dilution buffer. The proteins elution from the beads was done twice at room temperature with shaking, the first elution with 45µL 0.2M glycine pH 2 solution and the second elution with 45µL 0.2M glycine pH 2 (supplemented with 1%SDS) solution. Finally combined protein eluates were neutralized with 10µL 1M Tris-base pH 10. For proteomic analysis three biological replicates were generated.