Fluorescent Protein Knock-in
Fluorescent Protein Knock-in - Related Videos
08:46
Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli
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Cited by 34 •
2015
A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.
08:47
Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration
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Cited by 12 •
2012
We have developed a novel technique of quantifying nicotinic acetylcholine receptor changes within subcellular regions of specific subtypes of CNS neurons to better understand the mechanisms of nicotine addiction by using a combination of approaches including fluorescent protein tagging of the receptor using the knock-in approach and spectral confocal imaging.
06:26
Rapid Assessment of Membrane Protein Quality by Fluorescent Size Exclusion Chromatography
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Cited by 2 •
2023
The present protocol describes a procedure to perform fluorescent size exclusion chromatography (FSEC) on membrane proteins to assess their quality for downstream functional and structural analysis. Representative FSEC results collected for several G-protein coupled receptors (GPCRs) under detergent-solubilized and detergent-free conditions are presented.
09:17
Generation of Maternal Mutants Using zpc:cas9 Knock-in Zebrafish
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2025
Here, we describe a protocol for generating maternal mutant that couples a stable zpc:cas9 knock-in line with Tol2-mediated delivery of sgRNA expression cassettes.
03:41
Fluorescence Anisotropy-Based Detection of Protein-Protein Interactions
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2025
In this video, we describe the fluorescence anisotropy technique to study the interactions between the fluorophore-tagged Shwachman-diamond syndrome (SBDS) protein and the elongation factor-like 1 GTPase (EFL1). On incubating SBDS proteins with gradually increasing concentrations of EFL1, a steady increase in anisotropy is observed, indicating a successful interaction between the two proteins.
FAQs
Because the tag remains linked to the endogenous gene, fluorescence can reflect that gene’s native regulation rather than the behavior of an overexpressed reporter. This distinction helps investigators interpret cellular expression and protein location within the gene’s normal genomic context, supporting more reliable conclusions about neuronal biology and protein behavior.
The donor template carries the fluorescent sequence and provides the information needed for its incorporation at the selected genomic locus. After CRISPR-Cas9 creates a targeted DNA break, the cell can use homology-directed repair or a related precise editing mechanism to connect the tag with the endogenous gene.
The signal can show where a protein is located and how it is distributed within living neural cells. It can also support monitoring of protein trafficking, while the same labeling strategy helps researchers visualize neurons and follow structures such as axons and dendrites during studies of neural organization.
A typical workflow selects the genomic locus, uses CRISPR-Cas9 to create a targeted DNA break, and supplies a donor template containing the fluorescent sequence. Cellular repair then uses homology-directed repair or a related precise mechanism to place the tag at that locus. Microscopy is used afterward to examine fluorescence in living cells.
In neuroscience, the approach can label neurons, trace axons and dendrites, and examine protein trafficking or distribution with microscopy. These observations support research on neural circuits, development, and disease mechanisms. Linking the signal to the endogenous gene also helps relate neural structure and protein behavior to native genomic regulation.
It is most useful when researchers need observations that more closely reflect native regulation than an overexpressed reporter would. Connecting the fluorescent sequence to the endogenous gene preserves its relationship with the gene’s normal genomic context, making the method valuable for examining protein location, trafficking, or distribution in living neural cells.