Fluorescence Anisotropy-Based Detection of Protein-Protein Interactions

For fluorescence anisotropy-based protein-protein interaction detection, begin with recombinant target proteins tagged with C-terminal tetracysteine motifs in a suitable anisotropy buffer.

Add a fluorophore-containing dye — binding to target proteins via the tetracysteine motif. Dialyze with the anisotropy buffer, removing unbound dye. Transfer the mixture to quartz cuvettes. Measure the absorbance, determining the percentage of successfully-labeled target proteins.

In a fluorescence cuvette, mix the fluorophore-labeled target proteins with unlabeled proteins. Using a spectrofluorometer, measure fluorescence anisotropy.

As the target proteins suspend freely in the buffer, the fluorophores attached to them rotate randomly around their axes due to Brownian motion. Upon excitation with vertically-polarized light of an appropriate wavelength, the fluorophores emit light polarized in the vertical and horizontal planes. The ratio of the fluorescence intensities of the vertical and horizontally polarized emissions — anisotropy — is measured.

When unlabeled proteins bind to the fluorophore-labeled targets, the molecular size of the protein complex increases, reducing its rotational motion.