A representative repertoire of the VHH library was displayed on phage particles using M13KO7 helper phage infection (catalog number 170-3578 New England, BioLabs, UK). Three consecutive rounds of immuno-affinity selection were carried out on 96-well microtiter plates (catalog number M5785-1CS Sigma Aldrich, MO, USA) pre-coated with hTNC (1 μg/panning, O/N at 4°C). After each round of biopanning, bound phage particles were eluted (100 mM triethylamine, pH 10.0, catalog number T0886 Sigma Aldrich, MO, USA) and immediately neutralized with 1 M Tris-HCl, pH 7.4 (catalog number CE234 GeneON, Germany) and used to infect exponentially growing TG1 E. coli. Following the third round of biopanning, individual colonies were randomly picked. VHH expression was induced with 1 mM isopropyl-D-thiogalactopyranoside (IPTG, catalog number 2900245 5PRIME, Germany) in the periplasmic bacterial compartment. Solid phase ELISA of each periplasmic extract was carried out on hTNC (1 μg/mL), using a mouse anti-HA antibody (catalog number H9658 Sigma Aldrich, MO, USA) and goat anti-mouse IgG-peroxidase antibody (catalog number A9044 Sigma Aldrich, MO, USA).
Goat anti mouse igg peroxidase antibody
Manufactured by Merck Group
Sourced in United States
Goat anti-mouse IgG-peroxidase antibody is a secondary antibody that binds to mouse IgG and is conjugated with the enzyme peroxidase. It is used in various immunoassays and immunohistochemical techniques to detect and quantify the presence of mouse IgG in samples.
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Lab products found in correlation
2 protocols using goat anti mouse igg peroxidase antibody
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Nanobody Library Display and Screening
2
Western Blot Analysis of Fluorescent Proteins
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Sample preparation for western blot analysis was performed as in Denoncin et al.,83 (link) starting from 3 mL in the case of cleared B. bacteriovorus lysates. Sample were loaded on NuPage Bis-Tris SDS precast polyacrylamide gels and ran at 190 V for 50 minutes in NuPAGE MES SDS running buffer. Western blotting was performed using standard procedures with the following primary antibodies: JL-8 monoclonal antibody (Takara) for GFP variants, YFP and CFP; polyclonal mCherry antibody (product # PA5-34974, Thermo Fisher) for mCherry. Signal from antibody binding was visualized by detecting chemiluminescence from the reaction of horseradish peroxidase with luminol and chemiluminescence was imaged with an Image Quant LAS 500 camera (GE Healthcare). Goat anti-mouse IgG-peroxidase antibody (Sigma) was used as a secondary antibody for JL-8. Goat anti-rabbit IgG-peroxidase antibody (Sigma) was used as a secondary antibody for mCherry. Antibodies were diluted following manufacturer’s recommendations. Figures were prepared using ImageJ and assembled and annotated in Adobe Illustrator.
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