Nickel Affinity Column
Nickel Affinity Column - 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.
03:07
Column Chromatography - Concepts
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2020
Column Chromatography Multiple techniques exist to purify and separate compounds in an organic chemistry laboratory. One of the most reliable separation techniques for microscale separations, where less than 10 grams of the compound is available for analysis, is column chromatography. This technique separates compounds in a mixture based on their physical properties, such as their solubility, polarity, hydrophobicity, size, and charge. Chromatography Basics The main components of any...
09:15
Column Chromatography - Student Protocol
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2020
Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA Column Chromatography of Food DyeExpand In this experiment, you'll use column chromatography to separate green food coloring into its component blue and yellow dyes, called erioglaucine and tartrazine. First, you'll use 95% ethanol to elute the dye with a weaker affinity for silica gel. Then, you'll flush the second dye from the column with water. To recover the purified dyes, you will collect each...
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.
06:03
Purification of Self-Assembling Protein Nanoparticles using Affinity Chromatography
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2025
This video showcases the purification of histidine-tagged self-assembled protein nanoparticles or SAPNs using affinity liquid chromatography. The resulting purified SAPN fractions are suitable for vaccine development, holding promise for potential immunotherapeutic applications.
FAQs
Selectivity comes from coordination between immobilized nickel ions and histidine residues in the engineered polyhistidine tag. Proteins lacking the tag do not form the same binding interaction, so they are removed during washing while the tagged molecule remains associated with the column. This chemical recognition enables target enrichment from a complex cell lysate.
Imidazole releases the bound protein by competing with histidine residues for available nickel-binding sites. Adding it after washing changes the competition at the column, allowing the tagged target to leave the immobilized nickel while proteins that already passed through remain separated. The result is recovery of the engineered protein rather than continued retention on the column.
Washing removes proteins that have not formed the intended nickel-histidine interaction. Because the target remains associated through its polyhistidine tag, this stage improves separation before imidazole is introduced. The distinction is especially important when starting with complex lysates, where many cellular proteins are present and could otherwise accompany the recombinant molecule into later experiments.
The workflow uses a cell lysate as the starting material, followed by passage through the column, washing to remove non-target proteins, and imidazole addition to elute the retained tagged protein. Keeping these stages distinct separates binding from recovery and provides a practical route to obtaining purified material for immunology, infection, or structural studies.
Purified recombinant antigens can support antibody production and immune assays, where unwanted proteins from the original lysate could complicate interpretation. A Nickel Affinity Column provides a selective route to prepare these molecules before downstream use. The same purification approach can also supply other recombinant proteins needed for investigating immune recognition or related biological responses.
In infection research, purified recombinant enzymes and other proteins can provide defined material for examining pathogen biology. The method can also prepare proteins for structural studies, helping researchers work with a selected molecule rather than an unfractionated lysate. Its selective binding and straightforward workflow support reproducible preparation across experiments involving infectious-disease mechanisms.