Key Takeaways

  • Perform kinetic screening using high-specificity capture of His-tagged proteins

  • Benefit from low sample consumption to run experiments

  • Characterize either crude or purified nanobody samples on the LSA

Overview

The polyhistidine-tag is one of the most popular affinity tags for purification and detection of recombinant proteins. The histidine residues of the tag form coordination bonds with immobilized metal ions such as nickel, cobalt and zinc. This property is exploited for the stable capture of His-tagged protein. The NiHC200M biosensor is prederivatized with poly-Ni- NTA (nitrilotriacetic) groups for quick and easy immobilization of His-tagged molecules. In combination with the LSA instrument, the NiHC200M biosensor offers a fast and label-free approach for high throughput screening and quantitation of His-tagged protein libraries.

In this application note, we describe a generic approach for the kinetic screening of Histagged ligand using a NiHC200M biosensor. The presented protocol can be used as a starting point and can be adapted according to the properties of the sample.

Principle

The biosensor with pre-immobilized and nickel (Ni 2+ ) charged poly-NTA derivatized linear polycarboxylate hydrogel will specifically bind His-tagged ligand proteins. This approach can be adopted for the capture of purified or crude samples in quantitation and kinetic applications. Dissociation of the ligand and surface regeneration is achieved by the addition of a competitive ligand (imidazole) followed by the injection of a chelating agent (EDTA). Once the biosensor is regenerated, nitrilotriacetic groups are recharged with a solution of NiCl2 prior to the capture of the next set of ligand proteins.

Posted by Judicaël Parisot PhD

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