Real-Time Deubiquitination Assays Using a Free Ubiquitin Sensor
Yun-Seok Choi1, Robert E Cohen2
1School of Natural Sciences, Black Hills State University, Spearfish, SD, USA.
Methods in Molecular Biology (Clifton, N.J.)
|November 9, 2022
Summary
Researchers developed a new sensor, Atto532-tUI, to monitor deubiquitinating enzyme (DUB) activity in real time. This tool enables the study of DUBs using various ubiquitin conjugates, advancing deubiquitination assays.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Deubiquitinating enzymes (DUBs) are crucial for cellular processes, removing ubiquitin (Ub) from various substrates.
- Current DUB activity assays often rely on artificial substrates, limiting their applicability.
- Real-time monitoring of DUBs with native substrates is essential for accurate activity assessment.
Purpose of the Study:
- To develop a novel sensor for real-time detection of free ubiquitin release.
- To establish a versatile method for monitoring deubiquitinating enzyme activity using diverse ubiquitin conjugates.
- To present protocols for the preparation and application of the Atto532-tUI sensor.
Main Methods:
- Preparation of Atto532-tUI, a high-affinity sensor for free ubiquitin.
- Utilizing the sensor in real-time deubiquitination assays.
- Monitoring the release of free ubiquitin from various ubiquitin conjugates.
Main Results:
- The Atto532-tUI sensor effectively detects free ubiquitin release in real time.
- The developed assay allows monitoring of deubiquitinating enzyme activity with various ubiquitin conjugates.
- Protocols for sensor preparation and assay implementation are provided.
Conclusions:
- Atto532-tUI is a valuable tool for real-time deubiquitination assays.
- This method offers a versatile alternative to traditional DUB activity assays.
- The developed protocols facilitate the study of DUBs and their substrates.
