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Related Concept Videos

Tagging and Fusion Proteins01:24

Tagging and Fusion Proteins

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...
Reporter Genes02:11

Reporter Genes

Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.
Commonly used reporter...

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Related Experiment Video

Updated: May 23, 2026

Imaging Subcellular Structures in the Living Zebrafish Embryo
11:19

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Constructing and Expressing GFP Fusion Proteins.

David L Spector, Robert D Goldman

    CSH Protocols
    |April 10, 2012
    PubMed
    Summary
    This summary is machine-generated.

    This study presents a protocol for creating stable cell lines that express green fluorescent protein (GFP) fusion proteins. Stable expression simplifies cell cycle and biochemical analyses, offering advantages over transient expression methods.

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    Area of Science:

    • Cell Biology
    • Molecular Biology
    • Biochemistry

    Background:

    • Green fluorescent protein (GFP) fusion proteins are valuable tools for studying cellular and tissue-level biological questions.
    • While transient expression is rapid, stable expression in cell lines offers benefits like native expression levels and clonal isolation.
    • Stable cell lines facilitate advanced analyses such as cell cycle studies and biochemical fractionation.

    Purpose of the Study:

    • To provide a general protocol for the development of stable cell lines expressing GFP fusion proteins.
    • To highlight the advantages of stable cell lines for various biological investigations.

    Main Methods:

    • The protocol focuses on establishing stable transfection procedures for GFP fusion proteins.
    • It outlines a general transfection method applicable across various cell types, including HeLa, A-431, U2OS, BHK, and HT1080 cells.
    • Optimization of specific transfection techniques (e.g., electroporation, chemical methods) may be required depending on the cell line.

    Main Results:

    • The described protocol enables the generation of stable cell lines for consistent GFP fusion protein expression.
    • Stable expression allows for the generation of single-cell clones, mapping of integration sites, and determination of copy number.
    • Researchers can more easily perform cell cycle analyses and biochemical fractionations with stable cell lines.

    Conclusions:

    • Developing stable cell lines expressing GFP fusion proteins is a robust strategy for in-depth biological research.
    • This protocol offers a reproducible method for creating such cell lines, enhancing experimental capabilities.
    • Stable expression systems are crucial for accurate and comprehensive cellular and molecular analyses.