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Hi trap nhs columns

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HI-TRAP NHS columns are affinity chromatography columns designed for rapid and efficient purification of biomolecules. They contain N-hydroxysuccinimide (NHS) activated Sepharose resin, which allows for covalent immobilization of ligands for target molecule capture and purification.

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NHS) column (2 citations)

2 protocols using hi trap nhs columns

1

Affinity Purification of Antisera

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Antisera against MyoE and Uso1 were raised in rabbits by Davids Biotechnology (https://www.davids-bio.com). Animals were immunized with His6-tagged polypeptides containing residues 1082-1569 of MyoE (the GTD) or residues 1-659 of USO1. These polypeptides were purified by Ni2+ affinity chromatography after expression in E. coli BLB21 as described (Pinar et al., 2015 (link)). Antibodies against the target proteins were purified from raw antisera (40 mL) by affinity chromatography with the respective antigens, previously coupled to 1 mL HI-TRAP NHS columns (Cytiva #17-0716-01) packed with Sepharose pre-activated for covalent coupling of ligands containing primary amino groups, following instructions of the manufacturer. Antibodies were eluted with 100 mM glycine, pH 3.0, neutralized with 2M Tris, pH 7.5 and stored at −20°C.
Pinar M., Alonso A., de los Ríos V., Bravo-Plaza I., de la Gandara Á., Galindo A., Arias-Palomo E, & Peñalva M.Á. (2022). The type V myosin-containing complex HUM is a RAB11 effector powering movement of secretory vesicles. iScience, 25(7), 104514.
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2

Uso1 Antiserum Production and Purification

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Antiserum against Uso1 was raised in rabbits by Davids Biotechnology. Animals were immunized with the Uso1 GHD (residues 1–659), and tagged with His6x. Recombinant expression in E. coli and Ni2+ affinity purification are described below. Target antibodies were purified from raw antiserum by affinity chromatography through Hi-Trap NHS columns (#17-0716-01, Cytiva) charged with Uso1 antigen following the manufacturer’s instructions. Affinity-bound antibodies were eluted with 100 mM glycine (pH 3.0), then neutralized with 2 M Tris to a pH of 7.5 and stored at –20 °C.
Bravo-Plaza I., Tagua V.G., Arst H.N., Alonso A., Pinar M., Monterroso B., Galindo A, & Peñalva M.A. (2023). The Uso1 globular head interacts with SNAREs to maintain viability even in the absence of the coiled-coil domain. eLife, 12, e85079.
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