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Updated: May 21, 2026

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

A Protocol for the Identification of Protein-protein Interactions Based on ¹⁵N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Published on: September 24, 2012

Protein interaction affinity determination by quantitative FRET technology.

Yang Song¹, V G J Rodgers, Jerome S Schultz

¹Department of Bioengineering, Bourns College of Engineering, University of California at Riverside, 900 University Avenue, Riverside, CA 92521, USA.

Biotechnology and Bioengineering

|June 20, 2012

Summary

This summary is machine-generated.

This study validates Förster resonance energy transfer (FRET) as a sensitive and accurate method for determining dissociation constants (Kd) of protein-protein interactions, specifically within the SUMOylation cascade. The FRET assay provides reliable Kd values comparable to established techniques.

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Last Updated: May 21, 2026

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

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Identifying Protein-protein Interaction Sites Using Peptide Arrays

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Published on: November 18, 2014

Area of Science:

Biochemistry
Molecular Biology
Biophysics

Background:

The dissociation constant (Kd) is crucial for quantifying protein-protein interaction affinities.
SUMOylation, a key post-translational modification, involves complex protein interactions.
Förster resonance energy transfer (FRET) is a powerful technique for studying these interactions in vitro and in vivo.

Purpose of the Study:

To systematically evaluate and validate a FRET-based assay for determining the dissociation constant (Kd) of protein-protein interactions.
To assess the sensitivity and accuracy of the FRET method within the SUMOylation cascade.
To establish the general applicability of FRET for Kd determination.

Main Methods:

Utilized Förster resonance energy transfer (FRET) technology to measure protein-protein interaction affinities.
Employed the SUMO1 and Ubc9 interaction as a model system for assay development and validation.
Compared FRET-derived Kd values with those obtained from surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC).

Main Results:

The FRET-based method demonstrated high sensitivity and accuracy in determining the Kd of SUMO1-Ubc9 interaction.
Kd values obtained via FRET were in strong agreement with results from SPR and ITC.
Consistent results were observed across a FRET donor to acceptor concentration ratio range of 4-40.

Conclusions:

The FRET assay is a reliable and sensitive technology for determining dissociation constants (Kd) in protein-protein interactions.
This FRET-based approach is broadly applicable for quantifying binding affinities in various biological systems.
The study validates FRET as a robust tool for biochemical and biophysical research.