Researchers at the University of Birmingham, in collaboration with international institutions, have pioneered a groundbreaking new method for accurately counting labeled proteins in living cells. This innovative technique, known as Protein-tag Degree of Labelling (ProDOL), is set to revolutionize protein quantification in biological research.
The findings, published in a recent study, underscore the potential of ProDOL to significantly improve the precision and reliability of protein labelling in microscopy, making it an invaluable tool for biomedical research.
Fluorescence microscopy has been a fundamental technique in biomedical research for a significant amount of time, allowing researchers to identify and study proteins within cells. However, the precision of these analyses relies greatly on the capability to quantify the number of proteins that are tagged with fluorescent markers, which is referred to as the degree of labeling (DOL). Current approaches for assessing DOL have constraints, leading to inconsistent labeling effectiveness and non-specific signals.
“ProDOL is designed to address these challenges by providing a fast and reliable method to quantify labeling efficiencies,” said Professor Dirk-Peter Herten, the lead researcher at Cardiovascular Sciences, University of Birmingham. “This tool allows for the optimization of protein-tag labeling strategies and provides accurate measurements of protein copy numbers in cellular structures.”
“With its proven accuracy and adaptability, ProDOL is set to become a standard and valuable tool in the field of biomedical research, paving the way for more precise and insightful studies of cellular functions. It is also highly versatile, making it applicable to various cell types and experimental conditions.
“In the future, this could, for example, improve our understanding of cellular signaling processes in the activation of immune cells or platelets, which are relevant in the context of various diseases, including but not limited to inflammation, immune deficiencies, and various cardiovascular diseases.”
In the study, ProDOL was used to explore how the HIV-1 virus impacts the response of CD4 T immune cells. In collaboration with Professor Oliver Fackler from the Department of Infectious Diseases, Integrative Virology at Heidelberg University Hospital, the research demonstrated ProDOL’s efficacy in quantifying the total and activated copy numbers by counting both the total and active numbers of helper proteins in small signaling clusters of immune cells.
Additionally, the contribution of Professor Ursula Klingmüller from the Division of Systems Biology of Signal Transduction at the German Cancer Research Center (DKFZ) has been essential in designing probes for live-cell applications, ensuring the technique’s reliability and suitability for dynamic biological systems.
Journal reference:
- Stanimir Asenov Tashev, Jonas Euchner, Klaus Yserentant, Siegfried Hänselmann, Felix Hild, Wioleta Chmielewicz, Johan Hummert, Florian Schwörer, Nikolaos Tsopoulidis, Stefan Germer, Zoe Saßmannshausen, Oliver T. Fackler, Ursula Klingmüller & Dirk-Peter Herten. ProDOL: a general method to determine the degree of labeling for staining optimization and molecular counting. Nature Methods, 2024; DOI: 10.1038/s41592-024-02376-6
