Related Experiment Video
Updated: Jan 29, 2026
11:01
Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo
Published on: June 28, 2019
7.4K
Protein-Protein Affinity Determination by Quantitative FRET Quenching
Ling Jiang1,2,3, Zhehao Xiong1,2, Yang Song1,4,2
1Department of Bioengineering, University of California at Riverside, 900 University Avenue, Riverside, CA, 92521, USA.
Scientific Reports
|February 16, 2019
Summary
We developed a new method to measure molecular interaction affinity using fluorescence quenching. This approach complements existing techniques and offers broader applications for determining binding constants.
Area of Science:
- Biochemistry
- Biophysics
- Molecular Biology
Background:
- The dissociation constant (Kd) quantifies molecular interaction affinity.
- Quantitative Förster Resonance Energy Transfer (FRET) methods have been developed for Kd determination.
Purpose of the Study:
- To develop a novel method for Kd determination using donor fluorescence reduction via acceptor quenching in FRET.
- To validate this new method by comparing Kd values with established techniques.
Main Methods:
- Quantitative measurement of donor fluorescence quenching in FRET.
- Determination of Kd values for the SUMO1-Ubc9 interaction.
- Comparison with Kd values obtained from FRET acceptor emission and other technologies.
Main Results:
- A new Kd determination method based on donor fluorescence quenching was established.
- Estimated Kd values for SUMO1-Ubc9 interaction showed good agreement with other methods.
- The acceptor-quenched approach proved effective and complementary to acceptor excitation FRET.
Conclusions:
- The acceptor-quenched FRET method provides a reliable and versatile approach for Kd determination.
- This methodology is applicable whether the acceptor is a fluorophore or a quencher.
- These developments offer a comprehensive toolkit for assessing molecular interaction affinities in solution.
Related Concept Videos
02:26Protein Networks
4.5K
An organism can have thousands of different proteins, and these proteins must cooperate to ensure the health of an organism. Proteins bind to other proteins and form complexes to carry out their functions. Many proteins interact with multiple other proteins creating a complex network of protein interactions.
These interactions can be represented through maps depicting protein-protein interaction networks, represented as nodes and edges. Nodes are circles that are representative of a protein,...
These interactions can be represented through maps depicting protein-protein interaction networks, represented as nodes and edges. Nodes are circles that are representative of a protein,...
4.5K
02:04Protein-protein Interfaces
14.7K
Many proteins form complexes to carry out their functions, making protein-protein interactions (PPIs) essential for an organism's survival. Most PPIs are stabilized by numerous weak noncovalent chemical forces. The physical shape of the interfaces determines the way two proteins interact. Many globular proteins have closely-matching shapes on their surfaces, which form a large number of weak bonds. Additionally, many PPIs occur between two helices or between a surface cleft and a...
14.7K
01:22Protein-Drug Binding: Determination Methods
648
Determining protein-drug binding can be achieved through indirect and direct methods, each providing valuable insights into the interaction between proteins and drugs.
Indirect methods involve isolating the bound drug from its free form in biological samples such as blood, serum, or plasma. These techniques aim to measure the percentage of drugs bound to proteins. Equilibrium dialysis is a commonly used method where the free drug concentration at equilibrium is measured by separating the bound...
Indirect methods involve isolating the bound drug from its free form in biological samples such as blood, serum, or plasma. These techniques aim to measure the percentage of drugs bound to proteins. Equilibrium dialysis is a commonly used method where the free drug concentration at equilibrium is measured by separating the bound...
648
02:15Protein and Protein Structure
87.4K
Proteins are one of the most abundant organic molecules in living systems and have the most diverse range of functions of all macromolecules. Proteins may be structural, regulatory, contractile, or protective. They may serve in transport, storage, or membranes; or they may be toxins or enzymes. Their structures, like their functions, vary greatly. They are all, however, amino acid polymers arranged in a linear sequence.
A protein's shape is critical to its function. For example, an enzyme...
A protein's shape is critical to its function. For example, an enzyme...
87.4K
01:41Affinity and Avidity
38.7K
Overview
38.7K
02:26Conservation of Protein Domains Over Different Proteins
14.4K
Protein domains are small structurally independent units that are part of a single amino acid chain. Although these domains are often structurally independent, they may rely on synergistic effects to perform their functions as part of a larger protein. Protein domains may be conserved within the same organism, as well as across different organisms.
A limited set of protein domains often duplicate and recombine during evolution. These domains can be organized in different combinations to...
A limited set of protein domains often duplicate and recombine during evolution. These domains can be organized in different combinations to...
14.4K