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. As a result, the emitted light becomes more polarized, with higher emission in the vertical plane than the horizontal plane — increasing anisotropy.

Add increasing concentrations of the unlabeled protein and repeat the anisotropy measurement.

A non-linear increase in anisotropy with increased unlabeled protein addition indicates unlabeled protein binding at multiple non-identical binding sites on the fluorophore-tagged target proteins.

Mix 3 nanomoles of the SBDS-FlAsH protein with 3 nanomoles of the Lumio Green dye in a 5-microliter volume of anisotropy buffer. Let the reaction proceed for 8 hours at 4 degrees Celsius. After 8 hours, dialyze the sample against the anisotropy buffer overnight to remove the free dye. Measure the absorbance at 280 nanometers and 508 nanometers in a spectrophotometer using a quartz cuvette. Then, use the Lambert-Beer law to quantify the percent of labeled protein as described in the text protocol.

Before measuring the anisotropy value, each titration step of the protein-ligand should be done carefully, ensuring that the entire sample is dispensed into the solution, and becomes homogeneous.

In a fluorescence cuvette, place 200 microliters of 30 nanomolar SBDS-FlAsH in anisotropy buffer, and titrate 2 microliters of 30 micromolar EFL1. Mix thoroughly, and let the reaction stand for 3 minutes before measuring the anisotropy and fluorescence value. Repeat this process until a total volume of 40 microliters of EFL1 has been added. As a final step, fit the data to a presumed binding model, as described in the text protocol.