Selection between the two methods rests on determining whether the benefits of the higher overall signal outweigh the costs of a higher blank. The feasibility of simultaneous Quench and FRET analysis in PB-FRET makes it possible to identify not only sample autofluorescence in either or both channels, but also excitation quenching by the sample and emission quenching by the sample in either channel. TR-FRET data, while it excludes most autofluorescence, is less suitable for comparable analysis due to noisier data, particularly in Quench values. In addition to providing more robust data, PB-FRET offers logistic advantages as well. Since phycobiliproteins are extremely popular reagents in flow cytometry and other applications, and have been in use for years, hundreds of phycobiliprotein-conjugated antibodies are currently on the market, as well as activated pigments, conjugation kits and unconjugated phycobiliproteins, all at competitive prices.
Many vendors are also prepared to perform custom conjugations.
The conjugation technology is widely licensed and has no association with a particular equipment manufacturer. Conjugates with prepaid licensing are also available. In addition, the established market in phycobiliprotein conjugates helps to ensure reliable and cost- effective reagent availability. Table 3 summarizes the comparison of the two methods.