FCS Classroom
FCS Application: Protein-Protein Interactions
Fluorescence Correlation Spectroscopy (FCS) is an ideal technique for quantifying protein-protein interactions. FCS distinguishes components in your sample based on molecular weight, so it can easily differentiate bound protein-protein complexes from unbound proteins in solution.
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No Standard Curves
FCS measures the number of fluorescent particles in its detection volume, providing a direct measure of concentration without the need for standard curves. In addition to total concentration, FCS also gives you the relative concentration of complexed and free protein, so you will know exactly what concentration of your protein is bound and what concentration is unbound.
Measure Binding Kinetics
By measuring the change in the concentration of the bound protein-protein complex over time, you can use FCS to obtain information on binding kinetics (kon, koff) and binding affinities (Kd, Ka).
Analyze Competition/Inhibition
By measuring the decrease in concentration of protein-protein complex upon inhibitor/competitor addition, you can determine inhibitor binding affinities (Ki, IC50).
Obtain Stoichiometry Information
By measuring the change in particle number and/or molecular size, FCS can tell you the stoichiometry of each protein bound in the protein-protein complexes.
Create Multiplex Assays
If you use two different fluorescent labels, FCS can measure two separate interactions in a single sample.
No Immobilization Required
Unlike surface plasmon resonance (SPR), which requires immobilization, FCS allows you to measure protein-protein interactions of freely diffusing proteins in a more physiologically-relevant state.