Phycobiliprotein Dyes

Bright fluorescence and high stability

Benefits

• Strong light absorption and high fluorescence yields
• Absorption bands extend over a broad spectrum of the visible light 
• Excellent stability and highly fluorescent over a broad pH range
• High water solubility without the need to use organic solvents
• Compatible with common excitation sources and filter sets

Applications

• Labeling of biomolecules such as proteins, antibodies and nucleic acids
• Fluorescence microscopy
• Flow cytometry
• Fluorescence in situ hybridization (FISH)
• Fluorescence correlation spectroscopy (FCS)
• FRET assays
• Multicolor labeling

Phycobiliproteins are water-soluble proteins with covalently bound chromophores present in cyanobacteria and certain algae (rhodophytes, cryptomonads, glaucocystophytes) that capture light energy which is transferred to chlorophylls during photosynthesis. Phycobiliproteins can be used as fluorescent dyes to label (bio-)molecules to obtain specific probes or markers for highly sensitive cell and molecular biological applications such as immunofluorescence microscopy, flow cytometry, microarrays, studies of enzyme kinetics etc. The major phycobiliproteins are: R-Phycoerythrin, B-Phycoerythrin and Allophycocyanin. They show optimal quantum yields (up to 98%) and extinction coefficients as well as high resolutions and large stokes shifts. The fluorescence yield is equivalent to at least 30 unquenched fluorescein or 100 rhodamine molecules at comparable wavelengths (on a molar basis) and fluorescence is not quenched by external agents since it is protected by covalent binding to the protein backbone. They can be easily linked to antibodies - the sensitivity of antibodies conjugated with R-PE and B-PE is usually five to ten times greater than that of the corresponding fluorescein conjugate(s).

The red-fluorescent R-Phycoerythrin has a strong absorption peak at 565 nm, and a secondary absorption peak at 492 nm. The emission peak is at 575 nm. This multi-subunit protein is commonly used for fluorescent immunolabelling, particularly for applications involving fluorescence-activated cell sorting (FACS) or live cell staining. The absorption bands of R-Phycoerythrin extend over a broad spectrum of the visible light (green to far-red) including many common excitation wavelengths. This allows for versatility in the excitation source and enables large Stokes shifts, thus minimizing interference from Rayleigh-scattered light.

B-Phycoerythrin has a slightly different spectral characteristic than R-Phycoerythrin. It absorbs strongly at about 545 nm and emits strongly at 572 nm instead and might be better suited for some instruments. B-Phycoerythrin may also be less "sticky" than R-Phycoerythrin and contributes less to background signal due to non-specific binding in certain applications.

Allophycocyanin (APC), another member of the light-harvesting phycobiliprotein family absorbs red light at 650 nm and has a maximal emission at 660 nm. Allophycocyanin and its conjugates are both brighter and more photo-stable than e.g. CyÔ5 conjugates.

SMCC-R-PE* and SMCC-APC* are chemically activated (maleimide group from SMCC) for easy reaction with sulfohydryl (SH) groups such as reduced cysteine residues of proteins and antibodies. The activated R-PE/APC can be efficiently coupled to immuoglobulins to obtain highly fluorescent conjugates that are ideally suited for applications such as immunofluorescence microscopy or flow cytometry.

* Succinimidyl-4-(N-Maleimidomethyl)Cyclohexane-1-Carboxylate (SMCC) is a non-cleavable and membrane-permeable crosslinker containing an amine-reactive N-hydroxysuccinimide (NHS ester) and a sulfhydryl-reactive maleimide group. NHS esters react with primary amines at pH 7-9 to form stable amide bonds, while maleimides react with sulfhydryl groups at pH 6.5-7.5 to form stable thioether bonds.

** Upon dilution or exposure to chaotropic salts, APC reversibly disassociates. For example, one molar sodium perchlorate has been shown to be a particularly effective agent for demonstrating the disruption of the APC quaternary structure. This change in structure causes changes in both the absorption and emission spectra. APC decomposition can be prevented by the introduction of crosslinks between the alpha and beta subunits resulting in increased stability of cross-linked APC in the presence of a chaotropic salt compared to APC. Thus, cross-linking is well suited for maintaining the structural integrity of APC.

Note: Fluorescence Microscopy is also topic of various training courses of our PromoCell Academy! >More Information