Protocol for detecting interactions between intrinsically disordered proteins and long DNA substrates by
Alyssa Pastic1, Annahat Kochhar1, Damien D'Amours1
1Ottawa Institute of Systems Biology, Department of Cellular and Molecular Medicine, University of Ottawa, Roger Guindon Hall, 451 Smyth Road, Ottawa, ON K1H 8M5, Canada.
STAR Protocols
|July 24, 2025
Summary
This study introduces a new, radioactivity-free method to detect how intrinsically disordered regions (IDRs) of proteins bind to DNA. The protocol enables sensitive and reproducible quantification of these crucial molecular interactions.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Intrinsically disordered regions (IDRs) are protein segments lacking stable tertiary structures.
- IDRs are vital in cellular processes like molecular recognition and gene regulation.
- Understanding IDR interactions with DNA is key to elucidating their biological functions.
Purpose of the Study:
- To present a novel protocol for detecting intrinsically disordered region-DNA interactions in vitro.
- To establish a sensitive and reproducible method for quantifying these interactions.
- To offer a radioactivity-free alternative to existing assays.
Main Methods:
- Development of an electrophoretic mobility shift assay (EMSA) protocol.
- Utilization of long DNA substrates for enhanced detection.
- Implementation of a radioactivity-free detection system.
- Detailed steps for data analysis are provided.
Main Results:
- The protocol enables sensitive detection of IDR-DNA interactions.
- The method is reproducible, facilitating reliable quantification.
- The assay is free of radioactivity, enhancing safety and ease of use.
- Successful application demonstrated for characterizing IDR-DNA binding.
Conclusions:
- The presented protocol offers a robust and accessible method for studying IDR-DNA interactions.
- This technique aids in understanding the role of IDRs in biological systems.
- The radioactivity-free approach promotes wider adoption in research.
