Spike s1 s2 ecd his recombinant protein

A 5 p mol of recombinant human cathepsins B, K, L, S, and V (Enzo) was incubated with 5 ng/μl of SARS‐CoV‐2 (2019‐nCoV) Spike S1 + S2 ECD‐His Recombinant Protein (Sino Biological #40589‐V08B1) in phosphate buffer (pH 6.0), 2 mM DTT, 1 mM EDTA for at 37°C for defined time periods. Reducing SDS‐PAGE loading buffer was added and then heated at 95°C to terminate experiments. After SDS‐PAGE, protein was transferred to a nitrocellulose membrane (Bio‐Rad) and probed with SARS‐CoV/SARS‐CoV‐2 Spike Protein S2 Monoclonal Antibody (1A9) (ThermoFisher # MA5‐35946) and donkey anti‐mouse secondary antibodies tagged with an infrared fluorophore (Rockland Immunochemicals). Membranes were imaged with a LI‐COR Odyssey scanner.

The BIAcore T200 equipment (GE Healthcare, USA) and Amine coupling kit (GE Healthcare, USA) were used for immobilization of SARS-CoV-2 Spike S1 + S2 ECD-His recombinant protein (Sino Biological, China) in Series S Sensor Chips CM5 (GE Healthcare, USA). The detailed process is explained in the supplementary document.

Materials. SARS-CoV-2 (2019-nCoV) Spike S1 + S2 ECD-His Recombinant Protein, MERS-CoV spike antigen protein and Influenza AH1N1 (A/New Caledonia/20/1999) Hemagglutinin/HA-specific B cell probe (His Tag) were purchased from Sino Biological Inc. (China). DNA-aptamer sequences purchased from IDT Integrated DNA technologies (Leuven, Belgium) with HPLC purification were reported in Supporting Information file (Table S1). Purified Thrombin (THR), streptavidin from Streptomyces avidinii (SA), bovine serum albumin (BSA), Human Serum (from male AB clotted whole blood) and all powders for buffers were purchased from Sigma-Aldrich s.r.l. (Milan, Italy). The monoclonal anti-thrombin antibody produced in mouse was from Abcam (Cambridge, UK), the anti-mouse HRP-conjugated antibody was from Life Technologies and Cy3-Streptavidin (Cy3-SA) was from Zymed Laboratories (San Francisco, CA, USA). SuperSignal West Femto Chemiluminescent Substrate kit was purchased from Thermo Scientific (Rockford, IL USA) and 100 nm of gold evaporated on silicon wafer from Agar Scientific (United Kingdom) was utilized. M-dPEG®₈-Thiol and Biotin-dPEG®₃-Lipoamide for the self-assembled monolayer (SAM) were purchased from Stratech (United Kingdom).