The conditioned medium samples were thawed and vortexed for 20 s. The samples were spun at 4 °C and 14,000 rpm for 30 min. The supernatant was collected from the sample. For these samples, serum Albumin and IgG were removed using Thermo Scientific Albumin/IgG Removal Kit. Next, the depleted serum samples were concentrated and exchanged into 2-D Lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, and 30 mM Tris-HCl, pH 8.5). Protein concentration was measured in all samples using Bio-Rad protein assay method.
The reaction was stopped by adding 1.0 µL of 10 mM Lysine to each sample and incubating in the dark on ice for an additional 15 min. The labeled samples were then mixed. The 2X 2-D Sample buffer (8 M urea, 4% CHAPS, 20 mg/mL DTT, 2% pharmalytes, and trace amounts of bromophenol blue), 100 µL destreak solution, and Rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 20 mg/mL DTT, 1% pharmalytes, and trace amounts of bromophenol blue) were added to the labeling mix to make the total volume of 250 µL. We mixed well and spun before loading the labeled samples into the strip holder.
The reaction was stopped by adding 1.0 µL of 10 mM Lysine to each sample and incubating in the dark on ice for an additional 15 min. The labeled samples were then mixed. The 2X 2-D Sample buffer (8 M urea, 4% CHAPS, 20 mg/mL DTT, 2% pharmalytes, and trace amounts of bromophenol blue), 100 µL destreak solution, and Rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 20 mg/mL DTT, 1% pharmalytes, and trace amounts of bromophenol blue) were added to the labeling mix to make the total volume of 250 µL. We mixed well and spun before loading the labeled samples into the strip holder.