Histidine Tag Cleavage
Histidine Tag Cleavage - Related Videos
02:57
Inducible Expression of Histidine-Tagged Ricin Toxin A Chain in E. coli
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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 inducible expression of His-tagged ricin toxin A chain (RTA) in Escherichia coli. It outlines the steps involved in the selective culturing of bacteria transformed with a recombinant expression plasmid, induction of His-tagged RTA expression, and preparation of the bacteria for further...
07:09
Using Cleavage Under Targets and Tagmentation (CUT&Tag) Assay in Mouse Myoblast Research
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2024
Researchers new to the epigenetic field will find CUT&Tag a significantly easier alternative to ChIP assays. CUT&Tag has tremendously benefited the epigenetic studies on rare and primary cell populations, generating high-quality data from very few cells. This protocol describes performing H3K4me1 CUT&Tag assays on mouse myoblasts isolated from mouse hindlimb muscles.
01:33
Cleavage and Blastulation
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2019
After a large-single-celled zygote is produced via fertilization, the process of cleavage occurs while zygotes travel through the uterine tube. Cleavage is a mitotic cell division that does not result in growth. With each round of successive cell division, daughter cells get increasingly smaller. Zygotic Genome Activation At the beginning of embryogenesis, maternal mRNAs control development. However, by the eight-cell stage of cleavage, embryonic genes become activated in a process called...
09:17
Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay
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Cited by 3 •
2017
We report and demonstrate an optimized nitrocellulose binding assay that can be used to quantify autophosphorylation of purified bacterial histidine kinases. Our method has several advantages over traditional SDS-PAGE based techniques, providing a valuable alternative for characterizing these important proteins.
07:33
Physical Properties Of Minerals I: Crystals and Cleavage
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2023
Source: Laboratory of Alan Lester - University of Colorado Boulder The physical properties of minerals comprise various measurable and discernible attributes, including color, streak, magnetic properties, hardness, crystal growth form, and crystal cleavage. Each of these properties are mineral-specific, and they are fundamentally related to a particular mineral’s chemical make-up and atomic structure. This experiment examines two properties that stem primarily from symmetric repetition of...
FAQs
Recognition-sequence placement is the key design feature. The recombinant construct positions a protease recognition sequence between the polyhistidine tag and the target protein, allowing a site-specific protease such as TEV protease to act at that junction. This arrangement directs removal to the intended boundary and helps preserve the target protein’s native sequence after purification.
A purification tag can influence protein folding, activity, or molecular interactions, so retaining it may complicate interpretation of experiments. Removing the tag produces a protein closer to its native form, making subsequent measurements more representative of untagged behavior. This consideration is especially important when structural, binding, or functional properties are central to the study.
Nickel-affinity chromatography provides a post-cleavage separation step. The cleaved histidine-containing material and the protease can be separated from the processed preparation using their affinity for nickel, reducing unwanted components in the final sample. This step complements enzymatic processing by helping obtain a cleaner tag-free protein for downstream experiments.
The principal experimental difference is the presence or absence of a sequence that may affect folding, activity, or interactions. A retained tag can be useful during isolation, whereas the cleaved form more closely represents the native protein. Comparing these forms can help determine whether observed structural or functional behavior reflects the protein itself or the purification tag.
The workflow begins by engineering the recombinant protein with a protease recognition sequence between the His tag and the target sequence. After protein isolation, a site-specific protease, such as TEV protease, is applied to remove the tag. Nickel-affinity chromatography then separates the cleaved tag and protease, producing material suited to downstream analysis.
The approach requires a recombinant protein construct containing a polyhistidine tag and an intervening protease recognition sequence. It also uses a compatible site-specific protease, such as TEV protease, followed by nickel-affinity chromatography for separation. Together, these components connect construct design, enzymatic processing, and purification into a single tag-removal workflow.
It is useful when researchers need tag-free antigens, antibodies, or pathogen-derived proteins. Such proteins support structural studies, binding assays, and functional experiments in which a purification tag could affect interactions or activity. Removing the tag therefore helps align recombinant protein measurements more closely with the physiological behavior being investigated.
Tag-free pathogen-derived proteins can provide a more physiologically relevant basis for examining structure, binding, and function. In immunology, this may support studies of antigen or antibody behavior; in infection research, it can aid analysis of pathogen-associated proteins. The resulting data are less directly confounded by effects that the purification tag might introduce.