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Related Concept Videos

Affinity Chromatography01:03

Affinity Chromatography

Affinity chromatography is a powerful technique extensively utilized for separating and purifying specific biomolecules from complex mixtures. It capitalizes on the highly selective binding between an analyte and its counterpart, such as antibody-antigen interactions. The counterpart is immobilized on the stationary phase, forming an affinity column. The stationary phase typically consists of solid support, such as agarose or porous glass beads, immobilizing the affinity ligand. The mobile...
Extraction: Advanced Methods00:56

Extraction: Advanced Methods

Metal ions can be separated from one another by complexation with organic ligands–the chelating agent– to form uncharged chelates. Here, the chelating agent must contain hydrophobic groups and behave as a weak acid, losing a proton to bind with the metal. Since most organic ligands used in this process are insoluble or undergo oxidation in the aqueous phase, the chelating agent is initially added to the organic phase and extracted into the aqueous phase. The metal-ligand complex is formed in...
Complexation Equilibria: The Chelate Effect01:19

Complexation Equilibria: The Chelate Effect

In complexation reactions, metal atoms or cations interact with ligands to form donor-acceptor adducts called metal complexes. Ligands that bind through one donor site are monodentate, ligands with two donor sites are bidentate, and those with more than two donor sites are polydentate ligands. For example, ethylene diamine is a bidentate ligand that binds through two nitrogen donor atoms, forming a five-membered ring. EDTA is a polydentate ligand that binds through four oxygen and two nitrogen...
Ion-Exchange Chromatography01:09

Ion-Exchange Chromatography

Ion-exchange chromatography, or IEC, is a technique for separating ions based on their affinity for the stationary phase. The stationary phase is a cross-linked polymer resin with covalently attached ionic functional groups. The functional groups can be either positively charged (cation exchangers) or negatively charged (anion exchangers). A cation exchanger consists of a polymeric anion and active cations, while an anion exchanger is a polymeric cation with active anions. The choice of...
Metal-Ligand Bonds02:51

Metal-Ligand Bonds

The hemoglobin in the blood, the chlorophyll in green plants, vitamin B-12, and the catalyst used in the manufacture of polyethylene all contain coordination compounds. Ions of the metals, especially the transition metals, are likely to form complexes.
In these complexes, transition metals form coordinate covalent bonds, a kind of Lewis acid-base interaction in which both of the electrons in the bond are contributed by a donor (Lewis base) to an electron acceptor (Lewis acid). The Lewis acid in...
Complexometric Titration: Ligands00:43

Complexometric Titration: Ligands

Different monodentate and polydentate ligands are used as complexing agents in complexometric titration reactions. The formation of complexes by mono- and bidentate ligands involves two or more intermediate steps, limiting their use as complexing agents. In comparison, polydentate ligands can form complexes with metal ions in a single-step process, facilitating sharper end points. This means polydentate ligands, such as amino carboxylic acid derivatives, are most commonly employed in...

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Related Experiment Video

Updated: Jul 5, 2026

Quantification of Metal Leaching in Immobilized Metal Affinity Chromatography
05:35

Quantification of Metal Leaching in Immobilized Metal Affinity Chromatography

Published on: January 17, 2020

Metal-chelate affinity chromatography.

K J Petty1

  • 1Merck & Company, West Point, Pennsylvania, USA.

Current Protocols in Neuroscience
|April 23, 2008
PubMed
Summary

Purify histidine-tagged proteins using nickel-NTA resin via metal-chelate affinity chromatography. This method works for native or denatured proteins and includes renaturation and analysis protocols.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Chemistry

Background:

  • Recombinant proteins are crucial in biotechnology and research.
  • Efficient purification methods are essential for obtaining functional proteins.
  • Histidine-tagged proteins offer a convenient target for affinity purification.

Purpose of the Study:

  • To provide detailed protocols for expressing and purifying histidine-tagged fusion proteins.
  • To describe metal-chelate affinity chromatography using nickel-NTA resin.
  • To outline methods for protein renaturation, analysis, and resin regeneration.

Main Methods:

  • Expression of recombinant proteins with six consecutive histidine residues (His-tag).
  • Purification using immobilized metal-ion affinity chromatography (IMAC) with nickel-NTA resin.

More Related Videos

Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory
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Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory

Published on: August 23, 2018

Affinity Purification of a 6X-His-Tagged Protein using a Fast Protein Liquid Chromatography System
07:19

Affinity Purification of a 6X-His-Tagged Protein using a Fast Protein Liquid Chromatography System

Published on: April 26, 2024

Related Experiment Videos

Last Updated: Jul 5, 2026

Quantification of Metal Leaching in Immobilized Metal Affinity Chromatography
05:35

Quantification of Metal Leaching in Immobilized Metal Affinity Chromatography

Published on: January 17, 2020

Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory
08:02

Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory

Published on: August 23, 2018

Affinity Purification of a 6X-His-Tagged Protein using a Fast Protein Liquid Chromatography System
07:19

Affinity Purification of a 6X-His-Tagged Protein using a Fast Protein Liquid Chromatography System

Published on: April 26, 2024

  • Renaturation of denatured proteins via dialysis or solid-phase methods.
  • Analysis of purified protein and regeneration of NTA resin.
  • Main Results:

    • Successful purification of histidine-tagged proteins under native or denatured conditions.
    • Effective renaturation protocols for recovering protein function.
    • Established procedures for protein analysis and resin reuse.

    Conclusions:

    • Metal-chelate affinity chromatography with nickel-NTA resin is a versatile and effective method for purifying histidine-tagged recombinant proteins.
    • The provided protocols facilitate the expression, purification, renaturation, and analysis of these proteins.
    • This technique supports downstream applications in various biological and biotechnological fields.