Hrp conjugated anti his tag antibody

The purity and identity of the protein was evaluated on a15% gel with SDS–PAGE method. The gel was stained with Coomassie brilliant blue. Western blotting was done with anti-His tag antibody (Abcam, UK). For Western blotting, the gel was run at a constant voltage of 100 V for 45 min using a Mini Protein Tetra System (Bio-Rad, USA), in order to transfer the protein bands to the nitrocellulose membrane (CMG Company). The nitrocellulose membrane was then blocked with 5% skimmed milk in PBS-T for 16 hr at 4°C. The membrane was washed and detection was done with 1/5000 dilution of HRP conjugated anti-His tag antibody (Abcam, UK) and diaminobenzidine (DAB) (CMG Company) as substrate.

For phage-ELISA, the wells of 96-well black plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with 1000 ng of HER2-Fc, streptavidin (Wako, Osaka, Japan), or IgG1-Fc (R&D systems) and blocked with 2% Perfect-Block (MoBiTec, Göttingen, Germany) in PBS for 1 h. Phages (1 × 10¹⁰ pfu) were added to each well and incubated for 1 h. After washing the plate, the bound phages were detected with the anti-T7 fiber tail antibody (Merck Millipore) as a primary antibody and the anti-mouse IgG HRP-linked antibody (Cell Signaling Technology, Danvers, MA, USA).
For indirect ELISA, the wells were first coated with 50 ng of HER2-Fc and then blocked with 2% Perfect-Block in PBS for 2 h. After incubation with sample proteins for 1 h, the HRP-conjugated anti-His-tag antibody (Abcam, Cambridge, MA, USA) was incubated for 1 h with the samples and then treated with the QuantaRed Enhanced Chemifluorescent HRP Substrate kit (Thermo Fisher Scientific). The resulting fluorescence was measured using an Infinite F500 (Tecan, Mannedorf, Switzerland) with specific filters (E_x/E_m = 535 nm/590 nm).
For specificity evaluation, the wells were incubated overnight with HER2-Fc (50 ng/50 μL in PBS), EGFR-Fc (50 ng/50 μL in PBS; R&D systems), or 50 μL PBS, and applied to the indirect ELISA described above.