Kta explorer machine

IEX fractions of highest DC-suppressive activity were pooled for Gel filtration chromatographic fractionation. The gel filtration was performed on an Äkta Explorer machine (GE Healthcare) using a Superdex 200 10/300 column (GE Healthcare) equilibrated with 2 column volumes PBS (pH 7). Approximately 500 μl of 2.5 mg/ml protein solution was applied. Loading and elution was done in the same buffer using a flow rate of 0.4 ml/min. Protein elution was monitored by measuring the absorption at 280 nm and 400 μl fractions were collected. The eluted fractions were probed by Bicinchoninic acid assay for protein quantification before testing in the in vitro DC stimulation assay. The tested fractions were analyzed by SDS-PAGE. Immunosuppressive fractions (3) and non-active fractions (4) were lyophilized and the protein composition analyzed by LC-MS/MS.

The purity and structure of the antibodies were analyzed by Superdex 200 Increase 10/300 GL chromatography (GE Healthcare, Chicago, IL, USA) using methods previously established by our laboratory (31 (link)). The standard proteins Ferritin, Aldolase, Conalbumin, Ovalbumin, Carbonic anhydrase and Ribonuclease were used for calibration. A 500 μL sample mix containing the above proteins was loaded into the column and separated by the ÄKTA explorer machine (GE Healthcare, Chicago, IL, USA). For the antibody analysis, 100 μL of filtered antibodies 17 and 3G12 (2mg/mL) in 1 × DPBS (Dulbecco’s phosphate-buffered saline, Gibco, Waltham, MA, USA) were analyzed. Antibodies were eluted by DPBS buffer at a flow rate of 0.5 mL/min.

The purity and structure of the antibodies were analyzed by Superdex 200 Increase 10/300 GL chromatography (GE Healthcare, Chicago, IL, USA). The standard proteins used for calibration and their molecular weights were: Ferritin (M_r 440 kDa), Aldolase (M_r 158 kDa), Conalbumin (M_r 75 kDa), Ovalbumin (M_r 44 kDa), Carbonic anhydrase (M_r 29 kDa), and Ribonuclease A (M_r 13.7 kDa) at 3 mg/mL. A 500 μL sample mix containing above proteins was loaded to the column and separated by the ÄKTA explorer machine (GE Healthcare, Chicago, IL, USA). For the antibody analysis, 100 μL of filtered antibodies (2 mg/mL) in 1 × DPBS (Dulbecco’s phosphate-buffered saline, Gibco, Waltham, MA, USA) was analyzed. Antibodies were eluted by DPBS buffer at a flow rate of 0.5 mL/min.