Alexa fluor 647 n hydroxysuccinimidyl ester

Recombinant SARS-CoV-2 spike RBD proteins from WT (#40592-V08H) and Omicron (B.1.1.529; #40592-V08H121) strains were purchased from Sino Biological (Beijing, China) and labelled with AlexaFluor 647 as previously described (25 (link)). In brief, 100 μg of SARS-CoV-2 RBD protein was resuspended in H₂O and mixed with NaHCO₃ (pH 8.3) buffer to a final concentration of 100 mM. Dimethyl sulfoxide (DMSO)-reconstituted AlexaFluor™ 647 N-hydroxysuccinimidyl ester (Thermo Fisher Scientific, Waltham, MA, USA) was added at a threefold molar excess, and the reaction was incubated at room temperature for 1 h. The reaction mixture underwent size-exclusion chromatography in phosphate-buffered saline (PBS), pH 7.4, using an ÄKTA pure system and a Superdex 200 Increase 10/300 column (Cytiva, Marlborough, MA, USA) to separate the labelled protein from the free fluorophore. Labelled protein fractions were pooled and concentrated using an Amicon Ultra-0.5 10K centrifugal filter device (Millipore, Billerica, MA, USA) and glycerol was added to a concentration of 10% (w/v) prior to snap freezing in LN₂ and storage at −80°C. Upon thawing and before use, protein concentrations were quantified via Nanodrop using the molar extinction coefficient.