A trimeric immunoglobin G‐binding domain outperforms recombinant protein G and protein L as a ligand for fragment antigen‐binding purification

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“…Protein Labeling: The proteins were labeled with Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific, MA), 5(6)-carboxyfluorescein (FAM) (Sigma, MO), or Alexa Fluor 610 (Invitrogen, CA) according to our previous works [40] with some modifications. Briefly, the proteins were diluted to 1.5 mg mL −1 prior to addition to the dye at a molar ratio of 1:6 (protein:dye) and incubated in the dark for 1-2 h at room temperature.…”

“…The molecular weight and purity of the proteins were measured by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‒PAGE) and size‐exclusion chromatography (SEC) according to our previous descriptions. [ 40 ] The protein concentration was measured using a Protein DC kit (Bio‐Rad, CA). The purified proteins were dialyzed against phosphate‐buffered saline (PBS, 10 m m Na 2 HPO 4 , 137 m m NaCl, 2.68 m m KCl, 2 m m KH 2 PO 4 , pH 7.4) with the additional reducing agent Tris‐(2‐carboxyethyl)‐phosphine (TCEP, 1 m m ) overnight and stored at −80 °C for further use.…”

“…ABD‐Tri was conjugated to a prepackaged HiTrap NHS‐activated HP column (1 mL, GE Healthcare,Uppsala, Sweden) to produce an ABD‐Tri‐HP column according to our previous study. [ 40 ] For affinity chromatography, crude plasma was diluted with binding buffer (PBS plus 0.3 m NaCl) four times, followed by loading 600 µL plasma onto an ABD‐Tri‐HP column at a flow rate of 1 mL min −1 . After washing the column with 20 mL binding buffer, the bound proteins were eluted with glycine‐HCl (0.1 m , pH 2.5).…”