The most widely used method of fluorescence is FRET. In this method, the fluorescent donor protein is coupled to one of the proteins of interest and the acceptor to the other protein. If there is no interaction, only the fluorescence of the donor should be detected. However, if an interaction takes place (with both fluorescent proteins at a distance of 10 nm or closer), the fluorescent donor protein can excite the acceptor via resonance energy transfer (dipole-dipole coupling). It is normal that some emission of the acceptor is detected even without interaction. This happens because often a small portion of the excitation light is also able to excite the acceptor if there is a spectral overlap in the excitation of the two fluorescent proteins – even if suitable filters are used. Therefore, the FRET ratio (emission of the acceptor divided by emission of the donor) is normally used for quantification, with a significant increase in the ratio indicating an interaction. This type of FRET is sometimes referred to as Sensitized Emission FRET (SE-FRET or seFRET). However, since SE-FRET is the only common method used to measure FRET with a microplate reader, it is usually referred to simply as FRET. Other types of FRET used with fluorescence microscopes are FLIM FRET (see below) and FRET acceptor photobleaching (where the increase in fluorescence of the donor is measured after the photobleaching of the acceptor).
Like most methods based on fluorescence intensity, FRET suffers from a high background and therefore low signal-to-noise ratios, which limits its sensitivity. Several alternative methods have been developed to overcome this limitation: BRET (see below) and TR-FRET. In TR-FRET (time-resolved FRET), fluorophores with a very long fluorescence lifetime (mostly chelates of lanthanides, such as europium, terbium and samarium) are used to bypass interference from molecules with a short fluorescence lifetime or other factors (especially excitation light). Instead of measuring light when excitation light is on, TR-FRET measurements are performed hundreds of microseconds later. The chelates can still emit light, but all other fluorophores in the sample and of course the excitation light have already faded away. Time resolved fluorescence (TRF) is not to be confused with fluorescence lifetime (see below).
BiFC is a PCA that uses a cleaved fluorescent protein (GFP or another). Fragments of the protein are not fluorescent, but the fluorescent protein is reconstituted when both fragments are brought into close proximity. Similar to FRET, BiFC is a fluorescent method for measuring PPI, but there is one important difference: the complementation of the cleaved fluorescent protein fragments is irreversible, making the method unsuitable for investigating interaction dynamics. Another complication with this method is that the cleaved fragments eventually reassemble by themselves, which increases the detection of false positives and requires extreme care in planning the experiment and designing appropriate controls. However, the fact that BiFC is irreversible can become an advantage: It can be used to identify and quantify weak interactions that are difficult to investigate with other methods.