HEK293TN and hACE2-HEK 293 TN cells were grown at 90% confluency in 150 mm plates. Cells were then detached in PBS. After 3 washes, cells were resuspended in PBS buffer containing 20 μg/ml of recombinant SARS-CoV-2-RBD and incubated with agitation at 4 °C for 60’. Primary amine crosslinker Disuccinimidyl Suberate (DSS) (Thermo Fisher Scientific cat no. 21555) was then added to the mix at a 2 mM final concentration. Crosslinking reactions were performed at room temperature (RT) for 30 min in agitation and were then quenched by addition of 20 mM of TRIS pH 7.4 for 15’.20 (link) After two additional washes in PBS, cells were lysed in the following buffer: 50 mM TRIS pH 7.4, 150 mM NaCl, 1% TRITON and 1:100 protein inhibitor cocktail (Sigma–Aldrich cat no. P8340).
1 mg of total cell lysates were then incubated for 2 h at 4οC in constant rotation with protein G Dynabeads (Life technologies cat. No. 10004D) previously incubated with 25 μg/ml of anti-hACE2 antibodies (R&D systems, cat no. AF933). The beads were then washed three times with lysis buffer and proteins were eluted from the beads by incubation at 95 °C for 10 min with 2X Laemmli sample buffer. Immunoprecipitated samples were then loaded on separate gels for silver staining (to control Immunoprecipitation efficiency), coomassie blue staining (for subsequent mass spectrometry analysis) and western blotting.
1 mg of total cell lysates were then incubated for 2 h at 4οC in constant rotation with protein G Dynabeads (Life technologies cat. No. 10004D) previously incubated with 25 μg/ml of anti-hACE2 antibodies (R&D systems, cat no. AF933). The beads were then washed three times with lysis buffer and proteins were eluted from the beads by incubation at 95 °C for 10 min with 2X Laemmli sample buffer. Immunoprecipitated samples were then loaded on separate gels for silver staining (to control Immunoprecipitation efficiency), coomassie blue staining (for subsequent mass spectrometry analysis) and western blotting.