FRET | CFP/YFP and variants | Genetically encoded fluorescent proteins; labeling possible on intra- and extracellular side. | Relatively large size (27 kDa) and sometimes the XFP represents the same size as the target protein. |
| FlAsH/CFP | Small size of FlAsH (∼700 Da compared to 27 kDa for YFP); flexible positioning within target protein; less likely to perturb the protein function; similar spectral properties as YFP, thus same filters can be used for FlAsH and YFP; labeling possible on intra- and extracellular side. | Labeling procedure required; background staining, which can be reduced when using the 12-amino acid high-affinity motif for FlAsH; FlAsH is a bidentate ligand and thus may not be able to freely rotate, thus possible changes in fluorophore orientation should be kept in mind. |
FRET by sensitized emission | | | Emission cross-talk, excitation cross-talk, bleed-through. |
FRET by FLIM | mTurquoise instead of CFP | Mono-exponential fluorescence decay time. | |
Time-resolved FRET (TR-FRET) | Eu3+- and Tb3+-cryptate in combination with fluorescent protein. | Time-resolved recording, thus less background signal from autofluorescence; almost no orientation dependence of the fluorescent signal. | Labeling procedure required; chelating cage needs to be attached to specific label (antibody, substrate for SNAP-tag etc.); labels represent charged compounds, and thus the labeling is mostly restricted to the extracellular side; rather high costs for the labeling reagent compared to genetically encoded fluorescent protein. |
SNAP-tag | The SNAP-tag is a nonfluorescent adapter protein that can be used in combination with fluorophores for FRET. | Highly versatile tag that can be combined with several different color fluorophores and hence one cloned protein can be used in several ways without the need to clone different constructs. | Relatively large size (20 kDa) and sometimes the tag represents the same size as the target protein; labeling procedure required, only a very limited number of fluorophores used for labeling the SNAP-tag can cross the plasma membrane, thus mostly extracellular labeling achieved. |
CLIP-tag | | Modified SNAP-tag with different substrate specificity; can be used for orthogonal labeling. | |
BRET1 | RLuc and GFP (coelenterazine h) | No excitation by external light source required; low background. | Relatively large size (27 kDa) and sometimes the XFP represents the same size as the target protein; chemical substrate for RLuc needs to be added to generate bioluminescent light; some substrates for RLuc are short lived and permit only short time windows to be investigated; currently still rather low light intensity. |
BRET2 | RLuc, RLuc-2, -8, and GFP2 (DeepBlueC) | | |
BRET3 | RLuc8 and mOrange (coelenterazine) | | |