Ascorbate

A 4-fold molar excess of the spin label reagent MTSL (1-oxy-2,2,5,5-tetramethyl-D3-pyrroline-3-methyl) methanethiosulfonate (Toronto Research Chemicals) dissolved in acetonitrile was added to H3K36me3 peptide (residues 32-41) with a Gly33Cys substitution (SIGMA Genosys), dissolved in buffer [10 mM potassium phosphate (pH 6.8), 20 mM NaCl], and incubated at 25°C for 16 h in the dark. Unreacted MTSL was removed by using a PD MidiTrap G-10 column (GE Healthcare) equilibrated with distilled water. The spin labeled peptide eluted with water was lyophilized and dissolved in buffer, and the pH was adjusted to 6.8. A paramagnetic sample was obtained by adding MTSL-H3K36me3 peptide to ¹⁵N-labeled Eaf3 CD dissolved in buffer at a molar ratio of 1:1 (final concentration 0.3 mM), and the ¹H,¹⁵N-HSQC spectrum was recorded. A diamagnetic sample was generated by reducing the paramagnetic sample with a 3.2-fold molar excess of ascorbate (Wako) at 25°C for 4 h in the dark. After the reduction, the ¹H,¹⁵N-HSQC spectrum was recorded.

Gold nanoparticles with a 60 nm diameter (EMGC60) were purchased from BBI Solutions (Cardiff, UK). Three different recombinants—the S1-mFc recombinant protein (40591-V05H1, Sino Biological, Beijing, China), the S1 + S2 recombinant with His-tag (BSV-CoV-PR-40, BioServ, San Diego, CA, USA), and the RBD recombinant protein with an Fc tag (BSV-CoV-PR-09, BioServ) were purchased. As positive controls for neutralizing antibodies, the sIgA antibody (E-AB-V1027, Elabscience, Houston, TX, USA), IgG antibody (SPD-M180, Acro Biosystems, Tokyo, Japan), and IgG standard in the IgG ELISA kit (290-84201, FUJIFILM Wako, Osaka, Japan) were used. Anti-human IgA (A80-102A) and anti-human IgG (2049-01) were obtained from Bethyl Laboratories (Waltham, MA, USA) and Southern Biotech (Birmingham, AL, USA), respectively. Bovine serum albumin (BSA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Polyethylene glycol 20,000 (PEG) was obtained from Fluka. Luminol, trolox, ascorbate, trehalose dihydrate, and Bradford protein assay kits were purchased from FUJIFILM Wako.