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Related Experiment Video

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Imaging Subcellular Structures in the Living Zebrafish Embryo

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Published on: April 2, 2016

mBeRFP, an improved large stokes shift red fluorescent protein.

Jie Yang¹, Liang Wang, Fei Yang

¹Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan, Hubei, China.

|July 11, 2013

Summary

We developed mBeRFP, a novel red fluorescent protein that is brighter and more photostable than existing options. This new protein is ideal for advanced cellular imaging and tracking biological processes in real-time.

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Area of Science:

Biochemistry
Molecular Biology
Cell Biology

Background:

Fluorescent proteins are essential tools in biological research.
Existing red fluorescent proteins (RFPs) have limitations in brightness, photostability, and maturation time.
Large Stokes shift (LSS) fluorescent proteins offer advantages for multicolor imaging.

Purpose of the Study:

To develop a novel monomeric large Stokes shift (LSS) red fluorescent protein (mBeRFP).
To characterize the photophysical properties and performance of mBeRFP.
To demonstrate the utility of mBeRFP in cellular imaging applications.

Main Methods:

Generation and characterization of the mBeRFP fluorescent protein.
Comparison of mBeRFP with existing LSS-RFPs (mKeima, LSS-mKate2).
FRET-based detection of caspase 3 activity using mBeRFP and Alexa 647.
Dual-color live-cell imaging of mBeRFP and CFP during apoptosis.

Main Results:

mBeRFP exhibits excitation/emission peaks at 446/615 nm.
mBeRFP is approximately three times brighter than mKeima and LSS-mKate2.
mBeRFP demonstrates improved photostability, rapid maturation, extended lifetime, and monomeric nature.
A FRET pair of mBeRFP and Alexa 647 enables sensitive caspase 3 detection with a large Förster radius (6.5 nm).
Live-cell imaging successfully visualized Bax dynamics during cancer cell apoptosis using mBeRFP and CFP.

Conclusions:

mBeRFP is a superior monomeric LSS-RFP with enhanced brightness and photostability.
mBeRFP is suitable for FRET-based biosensing, such as caspase 3 activity detection.
mBeRFP is a valuable tool for multicolor live-cell imaging, particularly for studying dynamic cellular processes like apoptosis.