Gfp antibody

To perform AP-MS procedure, ∼20 × 10⁶ HEK293 cells were grown in two independent batches, representing biological replicates. After 24 hours of induction of protein expression using Doxycycline (1 μg/ml), cells were harvested. Cell pellets were lysed in high-salt NP-40 lysis buffer (10 mM Tris-HCl pH 8.0, 420 mM NaCl, 0.1% NP-40, plus protease/phosphatase inhibitors) with three freeze-thaw cycles. The lysate was sonicated as described in our previous reports (Marcon et al., 2014 (link); Schmitges et al., 2016 (link)). To remove genomic DNA and RNA, we treated cell lysates with Benzonase for 30 minutes at 4°C with end-to-end rotation. The resulting whole-cell extract was centrifuged to pellet cellular debris. GFP-tagged viral proteins were immunoprecipitated with 1 μg of GFP antibody (G10362, Life Technologies) overnight in the cold room followed by a 2-hour incubation with Protein G Dynabeads (Invitrogen). The beads were washed 3 times with high-salt buffer (10mM TRIS-HCl, pH7.9, 300mM NaCl, 0.1% NP-40) with end-to-end rotation in the cold room and twice with buffer without detergent (10mM TRIS-HCl, pH7.9, 100 mM NaCl). The immunoprecipitated proteins were eluted with 0.5M NH₄OH and lyophilized.