Nhs activated sepharose 4ff

Freund’s complete adjuvant (FCA), Freund’s incomplete adjuvant (FIA), rabbit anti-IgY peroxidase conjugate, Immobilon®-P (polyvinylidene difluoride, PVDF), a transfer membrane with pore size of 0.45 μm, 3¹,3¹,5¹,5¹-tetramethylbenzidine (TMB), nonfat dry milk and horseradish peroxidase-conjugated rabbit anti-chicken IgY were purchased from Sigma (USA). Polystyrene ELISA plates were purchased from Corning (USA). Amicon ultra-15 centrifugal filter devices were obtained from Millipore (USA). NHS activated Sepharose 4 FF were purchased from GE Healthcare (UK). Protein molecular weight markers were purchased from Takara (Japan). All the other reagents were of analytical grade.

cDNAs for heavy and light chains of Ra62 antibody [21 (link)] were subcloned into the pcDNA3.4 expression vector using PCR and In-Fusion Reaction (Clontech). Ra62 antibody was expressed using the Expi293F Expression System (Thermo Fisher Scientific), according to the manufacturer’s instruction. The antibody secreted in culture medium was purified by protein G Sepharose 4 Fast Flow (GE Healthcare), and then buffer was exchanged to PBS by PD-10 column (GE Healthcare). Antibody concentration was determined by extinction coefficient method [23 (link)] with NanoDrop spectrophotometer (Thermo Fisher Scientific). Purified antibody was frozen and stored at -30°C. Preparations of Ra62 antibody conjugated Sepharose (Ra62 antibody Sepharose) was conducted using NHS-activated Sepharose 4FF (GE Healthcare), according to the manufacturer’s instructions. Two mg of Ra62 antibody was immobilized on 1 mL of Sepharose. Ra62 antibody Sepharose was stored at 4°C.

cDNAs for the anti-AGIA antibody heavy and light chains [17 (link)] were subcloned into the pcDNA3.4 expression vector using PCR and In-Fusion Reaction. Anti-AGIA antibody was expressed using the Expi293F Expression System (Thermo Fisher Scientific) according to the manufacturer’s instruction. The antibody secreted in culture medium was purified by protein G sepharose 4 Fast Flow (GE Healthcare), and then buffer exchange was conducted using a PD-10 column. Purified antibody was frozen and stored at -20°C.
Preparations of antibody conjugated sepharose beads was conducted using NHS-activated Sepharose 4FF (GE Healthcare). Anti-AGIA antibody sepharose was stored at 4°C. HRP-conjugated antibody was prepared using the HRP Conjugation Kit (Abcam). Anti-AGIA antibody-HRP was stored at -30°C.