2.2. Instrumentation
A high pressure gradient AZURA Lab Bio LC system from Knauer
(Berlin, Germany) equipped with a biocompatible buffer pump P 6.1L
including two pump heads to perform a gradient between two buffers, a
single wavelength UV detector 2.1L with a 5-mm optical path flow cell, a
AZURA CM 2.1S conductivity monitor, and a multifunctional assistant
module for sample injection, column switching, and fractionation
collector was used for different experiments in this study. Clarity
chrome software was used for instrument control, data acquisition and
data processing. The purification steps were done by two XK 16 (16×400
mm, I.D) and XK 26 (26×600 mm, I.D) columns from GE Healthcare
(Illinois, USA). XK 16 is packed with Q-Sepharose XL virus licensed as
the first dimension and XK 26 is packed with Superdex 200 prep grade
(Sup-200) as the second dimension. Chromatographic runs in the first
dimension was performed based on anion exchange (flow rate 8 mL/min,
mobile phase A including 20 mM Tris-HCl, pH 7.3, mobile phase B
including 20 mM Tris-HCl, 500 mM KCl, pH 7.3), sample volume (120 mL by
feed pump loading), and the second column as size exclusion (flow rate
2.5 mL/min, mobile phase 20 mM Tris-HCl, 150 mM NaCl, pH 7.3), and
volume of injection (3 mL). The columns have circulating jacket and they
maintained at 4 °C temperature by a cooling system and chromatograms
were recorded at the wavelength of 280 nm. All buffers and samples were
filtered through 0.45 µm, Acrodisc® Syringe Filters, before
starting the experiments. The sanitization of the columns were performed
with 0.5 M sodium hydroxide for at least one column volume after
experiments and equilibrated with loading buffer. Finally, the columns
were stored in 20% (v/v) ethanol at the room temperature for long-time
storage.