Fluorescence polarization
Detect a small labelled ligand binding to a larger partner.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
1Tumbling, and the light it scrambles
A fluorophore absorbs light most strongly when its absorption dipole lies along the light's electric field, so polarised excitation picks out the molecules that happen to be aligned with it. If those molecules stayed still for the few nanoseconds before they emit, the emitted light would be polarised in the same direction.
2What sets the window and the KD
The window depends on the size difference between tracer and partner and on how rigidly the dye is attached: a dye on a long flexible linker spins on its own even when the peptide is bound, which compresses the window. The tracer concentration decides whether the midpoint measures the affinity.
Free tracer
Low mP, set by the tracer's size and the dye's mobility; a value outside the expected range is the first sign of trouble.
Bound plateau
High mP once the tracer is saturated; the window is bound minus free.
KD
Protein at half-saturation, when the tracer is well below KD; fitted with the quadratic equation otherwise.
Competition
Unlabelled ligands displace the tracer; the IC50 converts to a Ki with equations that account for tracer and protein concentrations.
3How it goes wrong
Because polarisation is a ratio of two channels, anything that adds light unequally to them moves the number. An uncalibrated G-factor shifts every well and can give the impossible result of negative mP.
Key terms
Practise these as flashcards →(I∥ − G·I⊥)/(I∥ + G·I⊥) × 1000: the excess of emission parallel to the excitation over perpendicular.
(I∥ − G·I⊥)/(I∥ + 2G·I⊥); it averages linearly over the species in a mixture, weighted by their intensity.
Correction for the instrument's unequal sensitivity to the two polarisations, set with a standard of known polarisation.
The small fluorescent ligand whose polarisation reports binding, used at a fixed low concentration.
The difference in mP between free and fully bound tracer; it sets the precision of the fraction bound.
The characteristic time for a molecule to tumble, which grows roughly with its volume; compared with the fluorescence lifetime it sets the polarisation.
Local rotation of a dye on a flexible linker, which keeps polarisation low even when the labelled molecule is bound.
A measure of screening assay quality from the window and the scatter of the positive and negative controls.
When to use it
- Measuring the affinity of a small labelled ligand, such as a peptide, oligonucleotide or small molecule, for a much larger protein in solution.
- High-throughput competition screens for compounds that displace a known ligand, in 384- or 1536-well plates with a simple mix-and-read format.
- Following binding at equilibrium without separating bound from free, for example to compare mutants of a peptide-binding domain.
- Situations where the labelled partner can be made small and the unlabelled partner is at least several times larger.
Compared with alternatives
Also solution-based and needs little material, but works whichever partner is labelled and does not need a large size difference; FP is faster and simpler for plate screens.
Label-free with ΔH and stoichiometry, and a good check that the label does not change the binding, but it needs far more protein and is slow.
Gives kinetics as well as affinity; prefer it when on- and off-rates matter or no small labelled ligand is available.
For protein–nucleic acid binding, separates complexes on a gel and can show several species; FP with a labelled oligonucleotide is quicker and truly at equilibrium.