Sepharose 4 fast flow

FF (10 mL) that was used for EV isolation was diluted 1:1 with DPBS to decrease the viscosity and then centrifuged at 300× g for 10 min, followed by centrifugation at 2000× g for 10 min. In order to remove apoptotic bodies, the sample was centrifuged at 20,000× g for 30 min, and then the supernatant was filtrated through a 0.2 µM syringe filter. The sample was concentrated down to 500 µl using Amicon^® Ultra-15 centrifugal filter units (10 kDa). EVs were isolated using size exclusion chromatography (SEC) benchtop columns (Econo-pac^® Disposable chromatography column, Bio-Rad, Berkeley, CA, USA) filled with cross-linked 4% agarose matrix of 90 µm beads (Sepharose 4 fast flow™, GE HealthCare Bio-Sciences AB, Uppsala, Sweden). Twenty fractions, each 500 µl, were collected, and the protein concentration of each fraction was determined with Bradford assay using Quick Start™ Bradford Protein Assay (Bio-Rad, Berkeley, CA, USA) according to the manufacturer′s protocol. Particle concentration in the fractions was measured using nanoparticle tracking analysis (NTA, ZetaView, Particle Metrix GmbH, Inning am Ammersee, Germany). Based on these analyses, fractions 5–7 (1.5 mL) were pooled together, concentrated with Amicon^® Ultra-15 centrifugal filter device (10 kDa cut-off) and were used in further experiments.

Normal human serum was depleted for hCC (dNHuS) and used to dilute the reference material in the replacement assays. Depletion was achieved using an affinity matrix prepared from mAb CyDI-1 and NHS-activated Sepharose^™ 4 Fast Flow (GE Healthcare). A Vivaspin 15R column (30 MWCO; Sartorius-Stedim, Göttingen, Germany) was used to concentrate 6 mg of the antibody and exchange the buffer.
Antibodies were coupled to 0.5 ml NHS-activated Sepharose according to the manufacturer’s recommendations for 1 h at room temperature. After overnight inactivation, the matrix was washed with NHS buffer C (0.1 M Tris-HCl, pH 8.5) and NHS buffer D (0.1 M acetate, pH 4) alternating six times. Prior to storage, we carried out one elution step with sterile glycine buffer (0.2 M glycine, pH 2.9) and a neutralisation step with sterile PBS.
For the depletion of 5 ml normal human serum, we carried out two incubation steps using the mAb CyDI-1 matrix, first using a batch approach (overhead mixing, 15 min, room temperature), followed by a gravity flow approach at room temperature. An intermediate elution step was carried out as described above.

Pull-down assay by using GM1-Sepharose for the toxins were performed as previously described [23 (link)]. Briefly, lyso-GM1 was coupled using NHS-activated Sepharose 4 Fast Flow (GE Healthcare, England) in 0.2 M NaHCO₃ and 0.5 M NaCl (pH 8.3) at room temperature for 4 h with rotation. After the coupling reaction, non-reacted groups on the Sepharose were blocked by 0.5 M ethanolamine in the coupling solution. Lyso-GM1 Sepharose was then washed with 0.1 M Tris–HCl, 0.1 M acetate and 0.5 M NaCl and resuspended with PBS in a 1:1 (volume/volume) ratio. GST-LD, GST-PD, GST, or CTxB was incubated with lyso-GM1 or lyso-GM1 non-coupling Sepharose (control Sepharose) in PBS for 2 h at 4 °C with rotation. After incubation, Sepharose was sedimented by centrifugation at 12,000×g. The supernatant was discarded, and the remaining Sepharose was washed twice with PBS. The bound proteins were then solubilized with sample buffer (62.5 mM Tris–HCl, 2% SDS, 10% glycerol, 0.001% bromophenol blue and 100 mM dithiothreitol) and boiled for 5 min. The sample was analyzed by SDS-PAGE and visualized by 0.5% Coomassie Brilliant Blue R-250.