Amicon pro purification system

The extract from S. integrifolium was subjected to DEAD Sephadex A-25 column (1.0 cm × 20 cm). The column was washed with dialysis buffer (0.1 M NaCl in 0.02 M Tris-HCl, pH 8.0) and a linear gradient of 0.02 to 1 M NaCl (flow rate 0.5 ml/min) purification was performed. The elution fractions were subjected to hemagglutination assay. Those fractions with hemagglutination activity were further applied to a Sephadex G-75 column (2.6 cm × 40 cm). The fractions containing hemagglutination activity were concentrated and dialyzed against phosphate-buffered saline (PBS) using an Amicon® Pro Purification System with a YM-10 membrane (Millipore Co., Billerica, MA, USA). The concentrated proteins eluted from Sephadex G-75 column were diluted into chitin gel binding buffer (50 mM Tris HCl, 1 mM EDTA, 500 mM NaCl, 0.1% Tween-20, pH 8) and loaded in a chitin gel column (New England Biolabs) equilibrated with binding buffer. After washing column, bound proteins were eluted with 20 mM acetic acid, and dialyzed in 5 mM sodium phosphate buffer, at pH 6.0. The fractions from chromatography were stored in −80 °C for electrophoresis.

Proteins from culture supernatants were filtered through 0.45 μm, then 0.2 µm pore sizemembrane filters, and the filtrates were precipitated by adding ammonium sulfate at a final concentration of 80% (w/v) and the suspension was kept at 4 °C overnight under gentle stirring. The precipitated proteins were collected by centrifugation at 22,000× g for 20 min at 4 °C and then dissolved in 25 mL phosphate-buffered saline (PBS) buffer (50 mM). The enzyme solution was dialyzed overnight in a 14 kDa cut-off dialysis tubing cellulose membrane at 4 °C against 500 mL of the same buffer, which was replaced four times every 2 h. The resulting solution was filtered again through 0.2 µm pore size membrane filters.
Anion-exchange chromatography was performed using a HiTrap Q HP 5 mL column (GE Healthcare, Little Chalfont, UK). The column was equilibrated with 50 mM PBS (buffer A). Bounded proteins were eluted by applying a linear NaCl gradient (0–1 M) in buffer A and fractions were collected at 1 mL.
Active fractions were subsequently concentrated using a 10 kDa cut-off Amicon Pro Purification System (Millipore, Burlington, MA, USA). Tubes were first washed with 50 mM PBS.
A second purification of the fractions with enzymatic activity was done by gel filtration using a HiLoad 16/60 Superdex 200 prepgrade column (GE Healthcare) in 50 mM PBS buffer. Fractions were collected at 1 mL.

Human immunoglobulins were purified and concentrated from serum using protein G Agarose (Millipore, ref. 16-266) and the Amicon Pro Purification System (Millipore, ref. ACS500024) with a 30kDa molecular weight cutoff Amicon Ultra Centrifugal Filter (Millipore, ref. UFC503024).
Normal human skeletal muscle myoblasts were cultured and nucleofected with purified immunoglobulins according to the protocol recommended by the supplier (Lonza) and using the P5 Primary Cell 4D-Nucleofector^™ X Kit L (Lonza, ref. V4XP-5024). Nucleofected cells were plated in differentiation medium for different numbers of days and harvested for RNA extraction and subsequent RNA sequencing 24 hours after unless otherwise indicated.