Figure 4
Evaluation of RBD-TMR Binding to ACE2. (A) RBD-TMR pull down and elution from the matrix. (His)6-tagged ACE2 was immobilized in an NTA matrix (300 μL of 0.9 μM ACE2 + 75 μL of agarose-NTA-Ni2+ resin). A control incubation was performed omitting ACE2. Samples were centrifuged and washed three times and subsequently incubated in the presence of 1 μM RBD-TMR for 30 min under mild agitation. Created with BioRender.com. (B) After centrifugation, the supernatant was transferred to a fresh tube and analyzed by fluorescence spectroscopy. (C) The matrix was washed three times and eluted with 300 μL of 20 mM Tris–HCl pH 8.0, 150 mM NaCl and 250 mM imidazole. The sample was centrifuged, and the supernatant was analyzed by fluorescence spectroscopy. In (B,C) black lines correspond to the incubation in the presence of ACE2, while red lines correspond to the controls without ACE2. The excitation wavelength was 550 nm, the bandwidths for both excitation and emission were of 5 nm. All the measurements were made at 25 °C.
