Amicon ultra 0.5 ml 3k centrifugal filters

SNA Sepharose (Glycodiag, Orléans, France) was washed thrice with 50 mM Tris-HCl, 150 mM NaCl buffer pH 7.5 (TBS) and CaCl2 1 mM. A 10-fold concentrated Ca-containing TBS buffer pH7.5 (500 mM Tris-HCL, 1.5M NaCl and CaCl2 10 mM) was diluted 1:10 in the 420 μL fraction containing the total purified IgGs and the solution was added to the SNA Sepharose gel and incubated overnight at 4°C. The gel was then centrifuged at 8,000 × g for 5 min. Di-sialylated IgGs were then eluted by adding 100 mM glycine pH 2.8 for 5 min and the eluate was recovered by centrifugation at 10,000 × g for 5 min and immediately neutralized at pH 7.4 with 1M Tris-HCl pH 9.0. A second elution was performed to ensure the recovering of total di-sialylated IgGs (23 (link)) bound to the gel. Both elutions were pooled and the protein content was estimated by spectrophotometry at 280 nm using nanodrop ND-1000 (Wilmingto, DE, USA). The flow-through (i.e., un/mono-sialylated fraction) and the elution fraction (i.e., di-sialylated fraction) for each purified patient serum were then dialyzed against PBS and concentrated on an Amicon Ultra-0.5 ml (3K) centrifugal filters (Millipore, Tullagreen, IRL) as recommended by the manufacturer. Each concentrated fraction was adjusted to the same final volume.