Phycobiliproteins are a family of photosynthetic light-harvesting proteins derived from microalgae and cyanobacteria. These proteins contain covalently attached linear tetrapyrrole groups, known as phycobilins, which play a critical role in capturing light energy. In microalgae and cyanobacteria, energy absorbed by these phycobilins is transferred efficiently via fluorescence resonance energy transfer (FRET), to chlorophyll pigments for their use in photosynthetic reactions. Because phycobiliproteins have extremely highfluorescence quantum yields and absorbance coefficients (also refered to as molar extinction coefficient) over a wide spectral range, they are incredibly fluorescent and an excellent choice for use in fluorescence applications, primarily flow cytometry.

Phycoerythrin (PE), allophycocyanin (APC) and their tandem conjugates are among the preferred phycobiliproteins for flow cytometry. Synthesis of these dyes to molecules having biological specificity (e.g. anitbodies, protein A or streptavidin) can be used in fluorescence-based detection applications that require high sensitivity but not photostability, such as fluorescence activated cell sorting (FACS) and immunophenotyping. Compared to organic and synthetic fluorescent dyes, advantages of phycobiliproteins as fluorescent labels include:

• Long-wavelength fluorescence excitation and emission to minimize auto-fluorescence from biological materials
• Minimal fluorescence quenching (phycobilins covalent bind to the protein backbone)
• Very high water-solubility
• Significant Stokes shifts with resolvable emission spectra for multicolor analysis
• Multiple sites for stable conjugation with organic and synthetic compounds such as antibodies, cyanine dyes oriFluor® dyes

Figure 1. Schematic diagram of a phycobilisome situated on the thylakoid membrane (A) and the energy transfer steps which include charge separation (B). Each phycobilisome contains a core consisting of allophycocyanins. From this core several rods made up of phycocyanin and phycoerythrin extend in an outward orientation and function as light harvesting antennae. Energy absorbed by these rods is efficiently transferred to the chlorophyll pigments for their use in photosynthetic reactions.