Sepharose 4b gel

Recombinant FXIII-A₂ (rFXIII) was a kind gift from Novo Nordisk (Måløv, Denmark). Human thrombin was purchased from CoaChrom (Maria Enzersdorf, Austria). Fetal bovine serum (FBS) was obtained from Life Technologies (Waltham, MA, USA). A very low residual amount of FXIII-A was detectable in FBS by a highly sensitive ELISA method [50 (link)]. In the medium containing 5% FBS, the FXIII-A concentration was 19.75 ng/mL, which corresponded to 0.2% plasma FXIII-A concentration. For certain experiments, FBS was depleted even from that low amount of FXIII-A by immuno-absorption chromatography using an anti-FXIII-A 3B2H12 mouse monoclonal antibody [50 (link)] that cross-reacts with bovine FXIII-A. The antibody was covalently bound to CNBr-activated Sepharose 4B gel (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), according to the protocol recommended by the manufacturer. After the depletion procedure, FXIII-A concentration of FBS was below the limit of detection.

Human serum albumins and glutaraldehyde solution (25% v/v) were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Anhydrous ethanol (>95%) was purchased from J.T. Baker Inc. (Phillipsburg, NJ, USA). Hydrogen tetrachloroaurate (III) hydrate (HAuCl₄·3H₂O) was purchased from Alfa Aesar (Haverhill, MA, USA). Sodium citrate dehydrate was purchased from Merck (Darmstadt, Germany). S-2-(4-Isothiocyanatobenzyl)-diethylenetriamine pentaacetic acid (p-SCN-Bn-DTPA) was purchased from Macrocyclics (Dallas, TX, USA). The ¹¹¹In-InCl₃ solution was obtained from the Institute of Nuclear Energy Research (Taoyuan, Taiwan). Cell culture dishes, flasks, and plastic ware were purchased from Corning Inc (Corning, NY, USA). Fetal bovine serum and cell culture medium were purchased from HyClone (Logan, UT, USA). Sepharose 4B gel and Poly-Prep chromatography columns were purchased from GE Healthcare (Chalfont St. Giles, UK) and Bio-Rad (Hercules, CA, USA), respectively.

Sepharose-4B coupled with antibody was prepared by a described method (36 (link)). Briefly, swelled and CNBr-activated Sepharose-4B gel (GE healthcare) was incubated with antibodies in coupling buffer (0.1 M NaCO₃ and 0.5 M NaCl, pH 8.0) overnight at 4 °C. Washing uncoupling antibodies and blocking nonspecific groups on the coupling antibody column were carried out in accordance with the manufacturer’s protocol.
Prepared anti-βγ-CAT and anti-FCGBP antibody-Sepharose-4B were packed into columns (1.56 × 8.3 cm) in accordance with the manufacturer’s protocol. Purified samples from anion exchanges were loaded onto the antibody-affinity column. After washing the column by equilibrated buffer and washing buffer at two concentrations of NaCl (100 and 200 mM), samples were eluted with elution buffer (50 mM Tris and 1 M NaCl, pH 8.0). The elution fractions were dialyzed and concentrated in PBS for experiments.