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Multimerized epitope tags for high-sensitivity protein detection.

R Steven Stowers¹

¹Department of Microbiology and Cell Biology, Montana State University, Bozeman, MT 59717, USA.

G3 (Bethesda, Md.)

|April 7, 2025

Summary

New epitope tag multimers dramatically increase protein detection sensitivity. This advance aids in visualizing low-expression proteins for gene function studies in model organisms like Drosophila.

Keywords:

Drosophila FlyBase epitope tag synaptic vesicle vGlut

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Area of Science:

Molecular Biology
Genetics
Neuroscience

Background:

Understanding gene function necessitates knowing protein localization.
Low-level protein expression poses challenges for antibody-based detection.
Existing epitope tag multimers have limitations in sensitivity.

Purpose of the Study:

To develop highly multimerized epitope tags for enhanced protein detection sensitivity.
To validate the efficacy of these novel tags in a model organism.

Main Methods:

Engineered tandem repeat multimers encoding up to 80X copies of epitope tags (V5, HA, MYC, FLAG, ALFA, OLLAS).
Created conditional vGlut alleles in Drosophila incorporating 40XV5 and 40XMYC tag multimers.
Utilized antibody detection for visualizing epitope-tagged protein localization and expression.

Main Results:

Demonstrated synaptic localization of epitope-tagged vGlut in Drosophila brain and neuromuscular junctions.
Achieved robust and easily detectable expression in presynaptic terminals, even in single neurons.
Verified conditional expression in specific subsets of adult brain neurons.

Conclusions:

Highly multimerized epitope tags significantly enhance protein detection sensitivity.
These novel tags are effective for visualizing low-expression proteins in vivo.
Facilitates antibody-based detection for a wide range of protein localization and expression studies.