Captureselect c tagxl affinity matrix prepacked column

The recombinant proteins were purified using a CaptureSelect^TM C-tagXL Affinity Matrix prepacked column (ThermoFisher) and followed the manufacturer’s guideline [41 (link)]. Briefly, The C-tagXL column was conditioned with 10 column volumes (CV) of equilibrate/wash buffer (20 mM Tris, pH 7.4) before sample application. Supernatant was adjusted to 20 mM Tris with 200 mM Tris (pH 7.4) before being loaded onto a 5-mL prepacked column per the manufacturer’s instructions at 5 mL/min rate. The column was then washed by alternating with 10 CV of equilibrate/wash buffer, 10 CV of strong wash buffer (20 mM Tris, 1 M NaCl, 0.05% Tween-20, pH 7.4), and 5 CV of equilibrate/wash buffer. The recombinant proteins were eluted from the column by using elution buffer (20 mM Tris, 2 M MgCl₂, pH 7.4). The eluted solution was concentrated and desalted with preservative buffer (PBS) in an Amicon Ultra centrifugal filter devices with a 50,000 molecular weight cutoff (Millipore, Burlington, MA, USA). The concentrations of the purified recombinant proteins were determined by the Bradford assay using bovine serum albumin (BSA) as a protein standard, aliquoted, and stored at −80 °C until use.

The recombinant proteins, named rS1Beta, were purified using a CaptureSelect C-tagXL affinity matrix prepacked column (Thermo Fisher), according to the manufacturer’s guidelines. Briefly, the C-tagXL column was conditioned with 10 column volumes (CV) of equilibration/wash buffer (20 mM Tris [pH 7.4]) before sample application. The supernatant was adjusted to 20 mM Tris with 200 mM Tris (pH 7.4) before being loaded onto a 5-mL prepacked column according to the manufacturer’s instructions at a rate of 5 mL/min. The column was then washed by alternating with 10 CV of equilibration/wash buffer, 10 CV of strong wash buffer (20 mM Tris, 1 M NaCl, 0.05% Tween 20 [pH 7.4]), and 5 CV of equilibration/wash buffer. The recombinant proteins were eluted from the column by using elution buffer (20 mM Tris, 2 M MgCl₂ [pH 7.4]). The eluted solution was concentrated and desalted with preservative buffer (PBS) in an Amicon Ultra centrifugal filter device with a 50,000-molecular-weight cutoff (Millipore). The concentration of the purified recombinant proteins was determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific) with bovine serum albumin (BSA) as a protein standard, and the proteins were separated by reducing SDS-PAGE and visualized by silver staining.