Minidawn treos 2 mals detector

To characterize the purity and oligomeric state of soluble coronavirus S and RBD, purified proteins were injected onto a SRT SEC-1000 column (4.6 × 300 mm, Sepax) at 0.35 mL/min in PBS (for S) or a Superdex 200 Increase column (3.2 × 300 mm, Cytiva) at 0.15 mL/min in PBS (for RBD). The column and the entire SEC-MALS system, which includes a 1260 Infinity II HPLC (Agilent), a miniDAWN TREOS II MALS detector (Wyatt) and a Optilab T-rEX refractive index detector (Wyatt), were equilibrated in PBS for at least 24 h prior to analysis. Molecular weights of S and RBD were determined using Astra (Wyatt).

ToxRp‐ox and ToxSp were purified according to the protocol described before and pooled in a 1:2 ratio with excess of ToxSp. To get rid of unbound ToxSp, the sample was further purified using a Superdex 200 Increase 10/300 column (GE healthcare). The resulting fractions were pooled and concentrated to 0.1 ml, containing 18 mg/ml of ToxRSp‐ox heterodimer using Amicon Ultra Centricons with a 3 kDa cutoff. The sample was loaded on a Superdex 200 Increase 10/300 column (GE healthcare) with a flow rate of 0.5 ml/min performed on a ÄKTA pure 25 (GE healthcare) connected to the miniDAWN Treos II MALS detector (Wyatt). The buffer used for the MALS‐SEC measurement contained 20 mM Tris, 150 mM sodium chloride, pH 7.5. The chromatogram resulted in a single peak eluting after 16.19 ml. The hydrodynamic radius determined by the MALS detector leads to a molecular weight of about 28 kDa, suggesting a heterodimer formation of ToxRSp‐ox.

Purified S proteins were analyzed on a SRT SEC-1000 column (5 μm, 4.6x300 mm, Sepax) at 0.35 mL/min in PBS running buffer. The SRT SEC-1000 column and SEC-MALS system, which includes a 1260 Infinity II HPLC (Agilent), a miniDAWN TREOS II MALS detector (Wyatt) and a Optilab T-rEX refractive index detector (Wyatt), were equilibrated in PBS at 0.1 mL/min for at least 24 hr prior to analysis. Molecular weights were determined using the Astra software package.