Related Experiment Video
Updated: Mar 24, 2026
06:54
A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues
Published on: June 3, 2021
4.7K
A Fluorometric Activity Assay for Light-Regulated Cyclic-Nucleotide-Monophosphate Actuators
Charlotte Helene Schumacher1, Heinz G Körschen2, Christopher Nicol3
1Institut für Biologie, Biophysikalische Chemie, Humboldt-Universität zu Berlin, Berlin, Germany.
Methods in Molecular Biology (Clifton, N.J.)
|March 12, 2016
Summary
Researchers developed a new assay to easily study light-controlled molecules that regulate cyclic-nucleotide-monophosphate (cNMP) levels. This tool speeds up the discovery and characterization of optogenetic actuators for cell biology and neuroscience research.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Optogenetics offers precise control over cellular functions using light-sensitive proteins.
- Sensory photoreceptors are key optogenetic tools for regulating cellular processes.
- Controlling intracellular cyclic-nucleotide-monophosphate (cNMP) levels is crucial for cellular signaling.
Purpose of the Study:
- To develop an efficient assay for discovering, engineering, and characterizing light-regulated cNMP actuators.
- To enable quantitative measurement of both cNMP synthesis and degradation.
- To facilitate research in optogenetics and second messenger systems.
Main Methods:
- Developed a fluorometric assay using the pH indicator 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF).
- Quantified proton release associated with cNMP formation and hydrolysis.
- Applied the assay to both nucleotide cyclases (e.g., bPAC) and phosphodiesterases (e.g., LAPD).
Main Results:
- The BCECF assay accurately measures cNMP metabolism.
- The assay is applicable to purified enzymes and crude cell lysates.
- Demonstrated utility with blue-light-activated bPAC and red-light-activated LAPD.
Conclusions:
- The BCECF assay accelerates the discovery and characterization of light-regulated cNMP actuators.
- This assay supports parallelization and automation for high-throughput screening.
- Facilitates advancements in optogenetics by enabling better understanding of cNMP regulation.
