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Protein Dynamics in Living Cells

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Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...

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Updated: Feb 17, 2026

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Published on: September 16, 2014

Protocols for Monitoring Unconventional Protein Secretion Using Luminescence and Trapping Approaches.

Eloïse Néel^1,2, Morgane Denus^1,2, William Fargues^1,2

¹IGF, Univ Montpellier, CNRS, Inserm, Montpellier, France.

Current Protocols

|February 16, 2026

Summary

This summary is machine-generated.

This study introduces a new quantitative method to measure unconventional protein secretion (UcPS) in mammalian cells. The developed toolkit enhances the analysis of UcPS pathways, crucial for cell communication and stress responses.

Keywords:

RUSH Retention Using Selective Hooks intracellular compartments protein trafficking secretory pathways split luciferase unconventional protein secretion

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Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Published on: May 26, 2014

Area of Science:

Cell Biology
Molecular Biology
Biochemistry

Background:

Unconventional protein secretion (UcPS) exports cytosolic proteins via non-canonical pathways, vital for cellular functions.
UcPS quantification is challenging due to low efficiency, high background, and difficulty distinguishing active secretion from leakage.
Recent advances like split luciferase reporters and RUSH system improve sensitivity and temporal control.

Purpose of the Study:

To present a sensitive, quantitative workflow for monitoring UcPS in mammalian cells.
To integrate NanoLuc split luciferase (HiBiT/LgBiT) complementation with RUSH system for enhanced UcPS analysis.
To provide a toolkit for dissecting UcPS mechanisms across various conditions.

Main Methods:

Developed a luminescence-based secretion assay using split NanoLuc (HiBiT/LgBiT) complementation.
Generated stable HiBiT reporter cell lines for sensitive UcPS detection.
Integrated siRNA-mediated gene knockdown, pharmacological perturbation, and RUSH system for mechanistic studies.

Main Results:

Established a robust, sensitive, and scalable workflow for UcPS quantification.
Demonstrated the utility of the toolkit for analyzing UcPS mechanisms.
Enabled synchronized cargo release and identification of trafficking intermediates using the RUSH system.

Conclusions:

The presented protocols offer a high-throughput toolkit for analyzing UcPS mechanisms.
This framework facilitates rigorous dissection of UcPS pathways in physiological and disease contexts.
The integrated approach enhances the study of essential cellular processes like intercellular communication and stress response.