Rabbit anti 6 his antibody

The expression of each WT-HBc, ΔHBc or Z_HER2-ΔHBc monomer was confirmed by western blotting. The purified WT-HBc, ΔHBc and Z_HER2-ΔHBc particles were analysed by SDS-PAGE and electro-transferred onto a nitrocellulose membrane. For the detection of the His6-tag, rabbit anti-6-His antibody (Bethyl Laboratories, USA) was used as a primary antibody at 1:1000 dilution for immunoblotting, followed by HRP-linked anti-rabbit (Cell Signalling Technology, USA) at 1:1000 dilution and Precision Protein StrepTactin-HRP Conjugate (Bio-Rad Laboratories, USA) at 1:10,000 dilution for secondary antibody. For the detection of the His6-tag, mouse anti-HBc antibody (Merck Millipore, USA) was used as a primary antibody at 1:1000 dilution for immunoblotting, followed by HRP-linked anti-mouse (Cell Signalling Technology, USA) at 1:1000 dilution and Precision Protein StrepTactin-HRP Conjugate (Bio-Rad Laboratories, USA) at 1:10,000 dilution for secondary antibody. The specific bands were detected with enhanced chemiluminescence (ECL) detection system. The membrane was imaged using the ChemiDoc™MP (Bio-Rad Laboratories, USA) and analysed with Image Lab (Bio-Rad Laboratories, USA) software.

For ELISA and immunoblotting tests, detection of M13 helper phage and M13-Nb was achieved using specific polyclonal antibody (33 ) and monoclonal antibody anti-M13 conjugated to horseradish peroxidase (HRP, GE Healthcare Life Sciences). For ELISA, detection of antigen-bound nanobodies was mostly accomplished using rabbit anti-6 × His antibody (Bethyl Laboratories Inc.) or with streptavidin–POD (Roche Life Science) when biotinylated nanobodies were used. Subsequent detection of rabbit or mouse antisera was completed using anti-rabbit or anti-mouse antibodies conjugated to HRP for ELISA tests or to alkaline phosphatase for immunoblotting (Bethyl Laboratories Inc.). For nanobody preparation, pMES4 phagemid and E. coli strains (TG1 and WK6) were kindly provided by Prof. S. Muyldermans (VUB, Brussels, Belgium). Plasmid constructs for expressing different GH antigens (TEV-GH, GFP-GH, and GFP) were prepared as previously described (32 (link), 34 (link)). Expression and purification of these antigens was performed in E. coli BL-21(DE3) Gold using standard protocol (32 (link)). Commercial un-tagged rhGH was obtained from sigma. d-Biotinoyl-ε-aminocaproic acid-N-hydroxysuccinimide ester (Biotin-7-NHS, Roche Life Science) was used to prepare biotinylated nanobodies via chemical bioconjugation according to the manufacturer’s instructions.

Expression level of hOCT2-myc-His and its mutated proteins on plasma membrane was measured using 2×10⁶ cells transfected with pcDNA3.1/hOCT2-myc-His and Pierce Cell Surface Protein Isolation Kit (Thermo Scientific) according to the manufacturer's protocol using rabbit anti-6His antibody (BETHYL Laboratories). Briefly, cell surface proteins were labelled with membrane-impermeable Sulfo-NHS-SS-Biotin reagent, and the treated cells were lysed. After the labelled membrane proteins were isolated with immobilized NeutrAvidin Agarose according to the manufacturer's instruction, the membrane proteins were eluted with SDS-PAGE sample buffer containing 50 mM dithiothreitol. Eluted proteins were analyzed by Western blotting after separation with 10.5% SDS-PAGE.