Nickel Affinity
Nickel Affinity - Related Videos
03:54
Nickel Affinity Chromatography-Based Protein Purification: A Technique to Purify Polyhistidine-Tagged Recombinant Proteins from Bacterial Cell Lysate
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2025
In this video, we demonstrate the nickel affinity chromatography technique to purify histidine-tagged pyrophosphokinase enzymes from Clostridium difficile bacteria.
08:40
Gyroid Nickel Nanostructures from Diblock Copolymer Supramolecules
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Cited by 3 •
2014
This article describes the preparation of well-ordered nickel nanofoams via electroless metal deposition onto nanoporous templates obtained from self-assembled diblock copolymer based supramolecules.
03:07
Electron Affinity
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2020
The electron affinity (EA) is the energy change for adding an electron to a gaseous atom to form an anion (negative ion). This process can be either endothermic or exothermic, depending on the element. Many of these elements have negative values of EA, which means that energy is released when the gaseous atom accepts an electron. However, for some elements, energy is required for the atom to become negatively charged, and the value of their EA is positive. Just as with ionization energy,...
01:41
Affinity and Avidity
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2019
Overview Antibodies bind to toxins or substances on the surface of cells, bacteria, viruses, or fungi. The substance is called an antigen, and the precise binding site is the epitope. The strength of the antibody-epitope interaction is called affinity. When an antibody binds an antigen by multiple epitopes, the cumulative strength of the interaction is called avidity. The strength of the interaction influences the elicited immune response. The Adaptive Immune System Increases Efficiency by...
02:54
An Affinity Chromatography Technique for the Purification of a Recombinant Bacterial Protein
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2025
Source: Becker, B., et al. A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast. J. Vis. Exp. (2017).This video demonstrates the purification of a polyhistidine-tagged recombinant protein using nickel-based affinity chromatography. It outlines key steps, including bacterial lysis by sonication, clarification and filtration of the lysate, and selective binding and elution of the target protein using imidazole.
FAQs
A polyhistidine tag presents multiple histidine residues that can coordinate with immobilized nickel ions. This repeated metal-binding interaction gives tagged recombinant proteins stronger affinity for the resin than many other components in a cell lysate. As a result, the tag provides a practical handle for separating the engineered protein from complex biological mixtures.
A chelator such as nitrilotriacetic acid anchors nickel ions within the affinity resin while leaving coordination sites available for binding groups on proteins. This arrangement keeps the metal associated with the solid support and creates the chemical basis for selective retention. Related chelators can serve the same supporting function in nickel-based purification systems.
Elution disrupts the interaction between the retained protein and immobilized nickel. Imidazole can compete with histidine-containing binding sites, while changing the binding conditions can also weaken the interaction. Because unbound proteins are removed before this step, controlled release produces an enriched fraction rather than returning the entire lysate to the collection.
The workflow begins with a cell lysate containing the recombinant protein, followed by passage over nickel affinity resin. Components that do not bind are removed during washing, and the retained target is then released by imidazole or altered binding conditions. Collecting the eluted fraction provides a protein sample enriched for the engineered molecule.
The essential components are a nickel-containing affinity resin, a chelator such as nitrilotriacetic acid or a related compound, and a recombinant protein carrying a polyhistidine tag. A cell lysate supplies the starting mixture, while imidazole or modified binding conditions supports elution. Together, these materials enable selective capture, washing, and recovery.
Nickel affinity purification can generate enriched recombinant protein samples for structural, enzymatic, and functional studies. The separation is therefore useful beyond obtaining a cleaner preparation: it supplies material for examining protein form, catalytic behavior, or biological activity. Its value comes from linking selective isolation with downstream analysis of the target molecule.