Related Experiment Video
Updated: Mar 31, 2026
Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations
Published on: September 17, 2012
Hyper sensitive protein detection by Tandem-HTRF reveals Cyclin D1 dynamics in adult mouse
Alexandre Zampieri1,2,3, Julien Champagne1,2,3, Baptiste Auzemery1,2,3
1CNRS, UMR-5203, Institut de Génomique Fonctionnelle, Montpellier, F-34094, France.
Insights
We developed Tandem-HTRF, a fast assay for detecting rare proteins in small samples. This method revealed unexpected expression patterns of Cyclin D1, a key cell cycle regulator, in adult organs.
Area of Science:
- Biochemistry
- Molecular Biology
- Assay Development
Background:
- Detecting low-abundance proteins is crucial for understanding cellular processes.
- Existing methods can be time-consuming or require large sample volumes.
Purpose of the Study:
- To develop a novel, rapid, and sensitive method for semi-quantitative detection of low-abundance proteins.
- To investigate the expression pattern of Cyclin D1 in adult organs using the new method.
Main Methods:
- Homogenous Time Resolved Förster Resonance Energy Transfer (HTRF) assay.
- Utilized lanthanide and d2/XL665 labeled antibodies targeting different epitopes of the same protein.
- Developed a "Tandem-HTRF" approach for enhanced sensitivity.
Main Results:
- The Tandem-HTRF assay can detect rare proteins from microliters of cellular lysate within one hour.
- Cyclin D1, a core cell cycle regulator, is sustained in adult organs.
- Cyclin D1 expression shows an unexpected pattern influenced by environmental challenges.
Conclusions:
- Tandem-HTRF is a sensitive and efficient method for studying sparse proteins.
- The findings highlight the dynamic expression of Cyclin D1 in adult tissues.
- This assay facilitates investigation of protein dynamics with limited biological material.
Abstract:
We present here a novel method for the semi-quantitative detection of low abundance proteins in solution that is both fast and simple. It is based on Homogenous Time Resolved Förster Resonance Energy Transfer (HTRF), between a lanthanide labeled donor antibody and a d2 or XL665 labeled acceptor antibody that are both raised against different epitopes of the same target. This novel approach we termed "Tandem-HTRF", can specifically reveal rare polypeptides from only a few microliters of cellular lysate within one hour in a 384-well plate format. Using this sensitive approach, we observed surprisingly that the core cell cycle regulator Cyclin D1 is sustained in fully developed adult organs and harbors an unexpected expression pattern affected by environmental challenge. Thus our method, Tandem-HTRF offers a promising way to investigate subtle variations in the dynamics of sparse proteins from limited biological material.
More Related Videos
13:15Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1
Published on: February 25, 2016
08:33Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis
Published on: December 5, 2017