biotinylated recombinant human ace2  (R&D Systems)

Journal: Journal of Molecular Biology

Article Title: Inhibition of SARS-CoV-2 Infection by Human Defensin HNP1 and Retrocyclin RC-101

doi: 10.1016/j.jmb.2021.167225

Figure Lengend Snippet: HNP1 and RC-101 destabilize SARS-CoV-2 Spike protein and inhibit its interaction with ACE2 (A, B) Binding of 50 nM Cy5-HNP1 to SARS-CoV-2 Spike (A) or BSA (B) was monitored by fluorescence anisotropy. Data from two independent experiments performed in duplicates is presented as mean ± SE. The K d s were calculated by fitting the data to the binding isotherm equation (see Methods). The x-axis in (A) indicates the Spike monomer concentration. (C, D) Stability of SARS-CoV-2 Spike protein in the absence and presence of HNP1 and RC-101 was assessed by DSF. (C) Data from three independent DSF experiments is presented as mean ± SE. (D) First derivatives of the fluorescence versus time traces are shown. Melting temperatures ( T m ) were calculated as the inflection points of the derivative curves; n/a – not applicable. (E) Precipitation of Spike by HNP1 and RC-101 was assessed by ultracentrifugation followed by SDS-PAGE of soluble (supernatant – S) and insoluble (pellet – P) fractions. (F) A schematic of Spike/ACE2 solid-phase binding assays. (G) Interaction of SARS-CoV-2 Spike with ACE2 in the absence and presence of HNP1 and RC-101 was tested in solid-phase binding assays. Data from three independent experiments is presented as mean ± SD. [Def.] is defensin concentration.

Article Snippet: Following washing, 2 μg/mL of biotinylated ACE2 (R&D Systems #BT933-020) in PBS was loaded (25 μL per well) and incubated for 1 h at 37 °C.

Techniques: Binding Assay, Fluorescence, Concentration Assay, SDS Page