Table 2. Summary of labeled-ligand methods discussed in this review.

$–$$$$ represents the cost of equipment, consumables, and protein sample.

Description	FA	MST	FRET	Radiolabeling
Mechanism	Polarized light is used to analyze the change in rotational time of molecules.	This method is based on measuring the movement of molecules in a temperature gradient.	Fluorescence measurements of donor/acceptor pairs in close proximity (energy transfer).	Radioactive labeled ligand is applied to detect its binding to a receptor.
Affinity range	nm–mm	pm–mm	nm–mm	nm–mm
Thermodynamics	Yesb	Yesb	No	No
Kinetics	Yesc	No	No	No
Sensitivitya	High	High	Medium	High
Advantages	The method is label-free compatible using intrinsic fluorescence. Small amount of sample.	Very low quantities are required. Label-free compatibility due to intrinsic fluorescence.	The use of fluorescence tags in both, receptor and ligand facilitate accurate concentration measurements.	Very useful in systems with limited amount of receptor and/or ligand due to high sensitivity. Compatible with membrane proteins.
Limitations	Small differences in size between the bound and unbound forms.	Hydrophobic fluorescent dyes can produce unspecific binding.	The FRET pair needs to be in close proximity for efficient energy transfer.	Requires the use of radioactive material.
Financial aspects	Requires fluorescence spectrophotometer with light polarizers.	Requires specialized equipment and capillaries and a protein labeling kit.	Requires a spectrophotometer with desired filters.	Requires radioactive labeling, specific facility and licensing.
	$$$	$$$	$$	$$

^aSensitivity is relative to the required sample concentration.

^bThermodynamic parameters can be obtained through the measurement of K_d at different temperatures.

^cThrough time-resolved fluorescence anisotropy.