His6 Tag Incorporation
His6 Tag Incorporation - Related Videos
09:27
Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins
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Cited by 9 •
2014
A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently tagged proteins simultaneously using the method of transient dominant selection is described here.
10:32
Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag
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Cited by 7 •
2012
A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.
11:26
High-throughput Gene Tagging in Trypanosoma brucei
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Cited by 21 •
2016
Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.
13:36
Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches
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Cited by 1 •
2015
Here, we present a protocol to generate a proof-of-principle divalent adenovirus type 5 (Ad5) vector Ad5/H5-HVR1-KWAS-HVR5-His6 by utilizing the Antigen Capsid-Incorporation strategy. This vector was demonstrated to exhibit qualitative fitness, the capability to escape Ad5-positive sera in vitro, and the antigenicity as well as immunogenicity to the incorporated antigens.
01:24
Tagging and Fusion Proteins
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2023
Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and...
FAQs
Selective capture depends on coordination between the histidine side chains and immobilized nickel or cobalt ions. When a recombinant protein carrying the sequence contacts the affinity material, these interactions retain the tagged species while other proteins are less strongly associated. This chemical selectivity provides the basis for enriching the desired protein before downstream immunological or biochemical analysis.
Imidazole can compete with histidine residues for the immobilized metal, promoting release of the bound protein. Altering pH can also disrupt the coordination interactions that support retention. Because either change affects binding strength, the selected elution condition influences how effectively the target is recovered from the affinity material and how suitable the resulting preparation is for later experiments.
The position of the six-histidine sequence can influence whether the tag remains accessible to the immobilized metal and whether it interferes with the protein itself. Tag placement may affect folding, activity, or accessibility of functional regions. These effects matter when a purified protein must preserve native-like properties for antigenicity studies, structural analysis, or functional testing.
A typical workflow begins with producing a recombinant protein that contains the six-histidine sequence, followed by exposing the protein preparation to immobilized nickel or cobalt ions. The tagged species is captured through coordination chemistry and then released with imidazole or altered pH. The recovered material can subsequently serve as a defined reagent for detection, assay, or analysis.
His6-tagged antigens, antibodies, and pathogen-associated proteins can support several research needs, including protein production, antigenicity studies, assay development, and structural or functional analysis. Their value comes from enabling preparation of defined protein reagents through affinity purification. This can help investigators examine immune recognition or pathogen-related protein properties using a more controlled material.
Purified tagged proteins can provide material for studying antigenicity, developing assays, and examining structural or functional properties. In infection research, pathogen-associated proteins can be prepared for focused investigation, while immunology studies may use antigens or antibodies as defined reagents. Interpretation should account for the possibility that tag placement or purification conditions influenced folding, activity, or accessibility.