Mp 2000

The
solution flow through the experimental apparatus is also shown in Figure 1. In this system,
the cation solution was pumped through 0.5 mm inner diameter tubing
(Pharmed) into the center channel (Ch-B) of the EAE at a rate of approximately
0.4 mL/min using peristaltic pump PP1 (MP-2000, ssl.eyela.co.jp).
The inlet of PP1 was connected to a selection valve (C25-3184EMH,
vici.com) to allow several different cation solutions to be selected.
Similarly, an anion solution was pumped into the cathode side (Ch-C)
of the EAE at 1.75–3.5 mL/min through 1.5 mm inner diameter
tubing (Pharmed) using another peristaltic pump (Gilson mini pulse
III, www.gilson.com) in conjunction
with another selection valve. Purified water (UPW) was pumped into
the anode side channel (Ch-A) via a Unimol pump (UPS-112E, www.nitto-kohki.co.jp) at
3.0 mL/min. The dc power for the EAE was supplied by a PA36-1.2B unit
(www.texio.co.jp) operating
in the constant current mode. The selection valves and dc power supply
were computer controlled.

Biologic duoflow chromatography system (Bio-Rad, USA) was utilized as the protein purification instrument especially in wash, elution, and clean step as described by Gu et al. (2016) with slight modification (Gu et al., 2016 ). A peristaltic pump was also utilized to apply the sample to the column. A ResourceQ column containing quaternary ammonium cation resin was chosen as the anion exchanger. Tris-HCl buffer (buffer A) was used as the equilibration buffer solution and Tris-HCl with 0.5 M NaCl (buffer B) was used as the elution buffer in ion exchange chromatography. Before applying the sample to the column, the sample was filtered by using mixed cellulose ester having pore and diameter of 0,45 μm and 47 mm respectively (ADVANTEC, Japan) to prevent any contaminant from entering the column. The sample was applied to the column using peristaltic pump (EYELA MP-2000, Japan). After connecting the column to the purification instrument, sample was washed by isocratic flow mode of 100% buffer A. The purification process continued by setting the elution process to gradient linear mode of 100%–0% buffer A and 0%–100% buffer B. The system was kept at – 4 °C with a flow rate of 3.0 mL/min. Most peaks were detected from 12 min to 26 min of the process for all samples. Tens of fractions were collected and ready to be grouped by SDS PAGE.