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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 efficiently transferred via fluorescence resonance energy transfer (FRET), to chlorophyll pigments for their use in photosynthetic reactions. Because phycobiliproteins have extremely high fluorescence quantum yields and absorbance coefficients over a wide spectral range, they serve as valuable fluorescent tags in a variety of fluorescence applications, primarily flow cytometry. Phycobiliproteins conjugated to molecules having biological specificity (e.g. immunoglobulin, protein A or streptavidin) are effective tools in fluorescence activated cell sorting (FACS), histochemistry, imaging and immunoassay applications.
Compared to organic and synthetic fluorescent dyes, phycobiliproteins offer several advantages when used as fluorescent probes. These advantages include:
- Intense long-wavelength excitation and emission profiles to minimize auto-fluorescence from biological materials
- Minimal fluorescence quenching contributed by the covalent binding of phycobilins to the protein backbone
- Very high water-solubility to facilitate chemical manipulation for conjugation reactions
- Significantly large Stokes shifts with resolvable emission spectra for multicolor analysis
- Multiple sites for stable conjugation with organic and synthetic compounds such as antibodies, cyanine dyes or iFluor™ dyes
Spectral Properties of Phycobiliproteins
There are two main classes of phycobiliproteins: phycoerythrins and allophycocyanins. These phycobiliproteins, which differ mainly in their protein structure, phycobilin content and fluorescent properties, are typically utilized in fluorescence analyses.
R-Phycoerythrin (2558) is a large 240 kDa phycobiliprotein isolated from red algae. It exhibits an intensely bright yellow-orange fluorescence with an extinction coefficient (ε) of 1,960,000 cm-1M-1 and a quantum yield (Φ) of 0.84. R-PE is excited by laser lines ranging from the 488 nm blue laser to the 561 nm yellow-green laser. Its absorbance spectrum, which is characterized by three absorption bands, shows a primary absorbance peak at 565 nm with two secondary peaks at 496 nm and 545 nm. The relative prominence of the secondary peaks varies significantly among PE’s derived from different species. R-PE has a fluorescence emission maximum at 575 nm, which is in the yellow-orange region of the visible spectrum. In comparison to Cy3® (141), which has an extinction coefficient (ε) of 150,000 cm-1M-1 and a quantum yield (Φ) of 0.24, R-PE is significantly brighter.
Figure 1. Absorption (left) and emission (right) spectra of ReadiUse™ PE (2500). PE can be efficiently excited by the 488 nm argon laser (blue), the 543 nm helium-neon laser (green) or the 561 nm yellow diode laser (green-yellow). PE can be visualized with a Cy3 filter set.
Allophycocyanin (2554) is a 105 kDA phycobiliprotein found in cyanobacteria and red algae. APC exhibits a bright far-red fluorescence with an extinction coefficient of 700,000 cm-1M-1 and a quantum yield (Φ) of 0.68. It is excited by red laser lines ranging from 633 nm to 647 nm, and has a primary absorbance maximum at 652 nm with a secondary maximum at 625 nm. APC has a fluorescence emission maximum at 662 nm, which is in the red region of the visible spectrum. In comparison to Cy5® (151), which also fluoresces in the red region, APC is significantly brighter and a much better choice for use in flow cytometry. This is due in part to the low extinction coefficient (ε = 250,000 cm-1M-1) and quantum yield (Φ = 0.20) of Cy5®.
Figure 2. Absorption (left) and emission (right) spectra of ReadiUse™ CL-APC (2503). APC can be efficiently excited by either the 633 nm or 647 nm red laser lines, and visualized with a Cy5 filter set.
R-PE and APC Products
We offer a complete portfolio of phycobiliproteins and their conjugates, including lyophilized R-PE and APC, tandem dyes, streptavidin conjugates, conjugation kits and more.
Lyophilized R-PE and APC
ReadiUse™ PE (R-Phycoerythrin) (2500) and ReadiUse™ CL-APC (Cross linked-Allophycocyanin) (2503) are supplied in a lyophilized form completely free of ammonium sulfate. Removal of this strong precipitating agent (ammonium sulfate) eliminates tedious protein purification processes such as dialysis, and facilitates the rapid conjugation of PE or APC to antibodies and other proteins such as streptavidin.
Tandem dyes consist of two different covalently linked fluorophores, a donor (e.g. R-PE or APC) and a longer-wavelength emitting fluorescence acceptor (e.g. Cy5®, Cy7® or iFluor™ 647). Through a process known as FRET, energy is transferred from an excited donor to the acceptor, which produces a fluorescence emission characteristic of the acceptor. In flow cytometry, tandem dyes are ideally suited for multicolor parametric analysis of cells due to their exploitation of a single excitation source and their significantly large Stokes shifts. For example, Figure 3 illustrates the excellent spectral separation of FITC (120), PE (2500), PE-Cy5® (2610) and PE-Cy7® (2616) that enables four-color analysis of cells without extensive compensation. Moreover, all four fluorophores are efficiently and simultaneously excited by the 488 nm argon ion laser.
Figure 3. Absorption and emission spectra of FITC (green), PE (yellow), PE-Cy5® (orange) and PE-Cy7® (red) illustrating their clear spectral separation that enables four-color analysis of cells. All four fluorophores are efficiently excited by the 488 nm argon laser line (blue). FITC can be visualized with a FITC filter set. PE can be visualized with a Cy3 filter set. PE-Cy5® and PE-Cy7® can be visualized with a Cy5 and Cy7 filter sets, respectively.