Anti human igg fc hrp conjugate

The CRM 197 protein (10 μg) was treated with 0.1 μg trypsin at 37ºC for 5 min and analyzed by 10% SDS-PAGE under reduced and non-reduced conditions. The protein bands were transferred to a nitrocellulose membrane (GE Healthcare, France). Then the membrane was incubated with 3F9 mAb (1 μg/ml) or mouse anti-CRM 197 polyclonal antibody (1:2,000 v/v) diluted in blocking solution (5% skim milk in 0.05% TBST), followed by anti-human IgG Fc-HRP conjugate (1:6,000 v/v, Thermo Fisher Scientific) or anti-mouse IgG Fc-HRP (1:5,000 v/v, Thermo Fisher Scientific), respectively. Finally, the bands were visualized using a chemiluminescent substrate (WEST-ZOL plus, iNtRON BioTechnology, Korea).

GST-preS1(1-56) was expressed in E. coli DH5a with 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 2 h, as described previously [19] . Cell lysates from uninduced or IPTG-induced cells and 10 μg of purified GST-preS1(1-56) were separated by 12% SDS-PAGE, transferred to a nitrocellulose membrane, and incubated with antibody (0.5 μg/ml, 10 ml), followed by antihuman IgG Fc-HRP conjugate (1:6,000 (v/v); Thermo Scientific, USA). The bands were visualized using a chemiluminescent substrate (WEST-ZOL plus, iNtRON BioTechnology, Korea).