PE and APC

Phycoerythrin (PE) and allophycocyanin (APC) are intensely bright phycobiliproteins isolated from photosynthetic organisms, such as algae and cyanobacteria. By maximizing both fluorescence quantum efficiency and molar absorptivity, PE and APC have the capacity to generate fluorescence signals far brighter than any commercially available synthetic fluorophore. When conjugated to molecules having biological specificity, such as antibodies, protein A or streptavidin, phycobiliproteins yield extraordinarily luminescent probes, that have found considerable success in fluorescence imaging applications that require high sensitivity but not photostability, primarily flow cytometry, FACS, immunophenotyping and ELISAs.

Phycoerythrin (PE)

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Phycoerythrin (PE) is a 240 kDa phycobiliprotein isolated from red algae. It exhibits an intensely bright yellow-orange fluorescence (~25-fold brighter than fluorescein) with a molar extinction coefficient of 1,960,000 cm^-1M^-1 and a fluorescence quantum yield of 0.84. The fluorescence spectra of PE in its native state is characterized by three absorbance peaks, a primary absorbance maxima at 565 nm and two secondary absorbance maxima at 496 nm and 545 nm, and a single emission maxima at 574 nm. This three absorbance maxima improves compatibility with imaging instruments equipped with blue (488 nm), green (532 nm) and yellow lasers (561-568 nm). Prior to using PE, it must first be conjugated to a molecule having biological specificity, such as an antibody, streptavidin or annexin V. PE and PE tandem conjugates are typically used for flow cytometry, microarrays and ELISAs, with limited uses in immunofluorescence microscopy due to its rapid photobleaching characteristics.

Advantages of PE

• Long-wavelength emission profiles minimizes autofluorescence 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
• 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 or iFluor^® dyes
• Tandem conjugates for multicolor flow cytometry - generate a range of emission signals with a single excitation laser

1. ε = molar extinction coefficient at their maximum absorption wavelength.
2. Φ = fluorescence quantum yield in aqueous buffer (pH 7.2).

Phycoerythrin Products

We offer a wide selection of PE products, including CD antibodies, secondary antibodies, streptavidin conjugates, labeling kits, PE tandem dyes and more. For expeditious labeling of PE to antibodies and other proteins use ReadiUse™ PE, a lyophilized version of PE free of ammonium sulfate buffers. The removal of ammonium sulfate eliminates tedious protein purification processes, such as dialysis, making for a quick and easy conjugation. Also available are PE tandem conjugates for multicolor flow cytometry using a singular laser source.

• CD antibody conjugates for immunophenotyping leukocyte populations and subpopulations
• Secondary antibody conjugates and tandems
• Streptavidin conjugates and tandems
• Stand-alone PE and tandem dyes for protein labeling
• PE and tandem dyes antibody labeling kits

 

Allophycocyanin (APC)

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Allophycocyanin (APC) is a 105 kDa phycobiliprotein found in cyanobacteria and red algae. It exhibits a bright far-red fluorescence with a molar extinction coefficient of 700,000 cm^-1M^-1 and a fluorescence quantum yield of 0.68. Compared to PE which has a larger extinction coefficient and quantum yield, APC is not as bright. The fluorescence spectral of APC in its native state is characterized by two absorbance peaks, a primary absorbance peak at 651 nm and a secondary absrobance peak at 625 nm, and a single emission maxima at 660 nm. This dual absorbance maxima improves compatibility with imaging instruments equipped with red lasers (633-647 nm). Just like PE, APC must first be conjugated to a molecule having biological specificity, such as an antibody, streptavidin or annexin V prior to using. APC conjugates are frequently used for flow cytometry, microarrays and ELISAs, with limited uses in immunofluorescence microscopy due to its rapid photobleaching characteristics.

Advantages of APC

• Long-wavelength emission profiles minimizes autofluorescence 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
• 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 or iFluor^® dyes
• Tandem conjugates for multicolor flow cytometry - generate a range of emission signals with a single excitation laser

1. ε = molar extinction coefficient at their maximum absorption wavelength.
2. Φ = fluorescence quantum yield in aqueous buffer (pH 7.2).

Allophycocyanin Products

We offer a wide selection of APC products, including CD antibodies, secondary antibodies, streptavidin conjugtes, labeling kits, APC tandem dyes and more. For expeditious labeling of APC to antibodies and other proteins use ReadiUse™ APC, a lyophilized version of APC free of ammonium sulfate buffers. The removal of ammonium sulfate eliminates tedious protein purification processes, such as dialysis, making for a quick and easy conjugation. Also available are APC tandem conjugates for multicolor flow cytometry using a singular laser source.

• CD antibody conjugates for immunophenotyping leukocyte populations and subpopulations
• Secondary antibody conjugates and tandems
• Streptavidin conjugates and tandems
• Stand-alone APC and tandem dyes for protein labeling
• APC and tandem dyes antibody labeling kits

 

PE and APC Tandem Dyes

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PE and APC 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). In this conformation, the excitation of one fluorophore (termed the "donor") results in the transfer of energy to, and the fluorescing of, the other (termed the "acceptor"). This transfer of energy from donor to acceptor occurs through Förster resonance energy transfer, or FRET. 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. Figure 5 illustrates the excellent spectral separation of FITC, PE, PE-Cy5^® and PE-Cy7^® 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.

Recommendations for successful tandem dye usage

• Avoid extended exposure to light, tandem dyes are high susceptible to photobleaching and should be protected from light at all times.
• Do not freeze tandem conjugates, this can result in the denaturing of the donor
• Be cautious, fixation and permeabilization steps can reduce tandem dye brightness
• Use appropriate controls, such as isotype controls for background, unstained cells and single color controls for compensation when performing multicolor flow cytometry
• Determine if compensation is necessary before analyzing data, this is due to two or more tandems with overlapping emission spectra

Product Ordering Information

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PE, APC, PerCP and Tandem Dyes Labeled to CD Antibodies

The following table outlines the fluorescence properties of available phcoerythrin (PE), allophycocyanin (APC), PerCP and tandem dye labeled anti-human CD antibodies for use in flow cytometry (FACS). Phycobiliproteins are uncharacteristically bright due to their high molar extinction coefficients and quantum yields, an enviable quality when imaging low-abundance targets. However, since phycobiliprotiens photobleach rapidly, they are not recommended for microscopy. For additional information on phycobiliprotein-labeled CD antibodies and availability of other clones click on any label in the table below.

1. ε = molar extinction coefficient at their maximum absorption wavelength (Units = cm^-1M^-1).