G3000sw

The samples obtained by ion-exchange chromatography were concentrated using Amicon Ultra-15 10 K filters (Merck Millipore, Burlington, MA, USA) and gel-filtration chromatography (TOSOH G3000SW) connected to high-pressure liquid chromatography (HPLC) (Hitachi L-6200) for further purification (flow rate: 0.5 mL/min). Elution fractions (0.5 mL/fraction) from gel-filtration chromatography were used to perform CBB staining and immunoblotting after resolving the proteins by SDS-PAGE as described earlier.

Samples obtained from ion-exchange chromatography were concentrated using Amicon Ultra-15 10K filters (Millipore) and subjected to gel-filtration chromatography (TOSOH G3000SW) using high performance liquid chromatography (HPLC; Hitachi L-6200) for further purification (flow rate: 0.5 mL/min). Elution fractions (0.5 mL/fraction) from gel-filtration chromatography were used to perform CBB staining and immunoblotting after resolving the proteins via SDS-PAGE as described above.

The purity of IgG samples was assessed using size exclusion high‐performance liquid chromatography (SE‐HPLC) and SDS‐PAGE as previously described (O'Riordan, Kane, et al., 2014; O'Riordan, Gerlach, et al., 2014). In brief, SE‐HPLC was performed using a TSK‐Gel column (G3000SW, Tosoh Biosciences LLC, Tokyo, Japan) eluted with 25 mm NaH₂PO₄ and 100 mm Na₂SO₄, pH 7.0.
Immunoglobulin G samples were electrophoresed using precast 4%–12% Bis‐Tris gels. All samples were diluted 1:10 with LDS sample buffer, and 5 μg IgG was loaded into each well. A molecular mass ladder and an IgG standard were run alongside the purified IgG samples. Gels were resolved at 200 V constant and variable current for 50 min using MOPS buffer in the inner chamber and MOPS buffer containing 0.25% antioxidant was used in the outer chamber. Protein bands were visualized within the gels using Coomassie‐based SafeStain following the manufacturer's procedure.