Streptavidin phycoerythrin, also known as SA-PE or R-PE streptavidin, is typically used for flow cytometry, microarrays, ELISA, and other applications that require either high sensitivity or simultaneous multicolor detection. Phycoerythrin is a member of a family of proteins called phycobiliproteins, which are derived from cyanobacteria and eukaryotic algae and exhibit extremely bright fluorescence and high quantum yields.
In practical applications, the sensitivity of streptavidin phycoerythrin conjugates is usually 5 to 10 times greater than that of the corresponding fluorescein streptavidin conjugate. In addition to streptavidin phycoerythrin conjugates, Life Technologies™ also offers conjugates of B-PE and allophycocyanin streptavidin. Advantages of using the streptavidin phycoerythrin and other phycobiliprotein–streptavidin conjugates include the following:
- Intense long-wavelength excitation and emission to provide fluorescence free of any interference
- Large Stokes shifts with extremely high emission quantum yields, which translate to very bright fluorescent molecules
- Fluorescence not quenched by external reagents, due to protection of the fluorophore by the protein backbone
- Very high water solubility
- Homogeneous structure with defined molecular weights
Molecular Probes® streptavidin R-PE conjugates are purified by HPLC to remove most aggregates and free R-phycoerythrin (Figure 1), minimizing background and improving the overall signal-to-noise ratio. Our HPLC methods also allow us to achieve high levels of lot-to-lot consistency, which means highly reproducible results for your experiments for either flow cytometry or microarrays (Figure 2).
For more detailed information on the spectral characterization of streptavidin phycoerythrin, see The Molecular Probes Handbook Phycobiliproteins—Section 6.4.
|Figure 1. Analytical size-exclusion chromatograms of free streptavidin (red curve, detected by absorption at 280 nm) and streptavidin phycoerythrin (blue curve, detected by absorption at 565 nm), demonstrating that the R-phycoerythrin conjugate is substantially free of unconjugated streptavidin.|
|Figure 2. Streptavidin phycoerythrin used to detect DNA on a microarray. Streptavidin phycoerythrin used to detect DNA on a microarray. A DNA microarray containing a decreasing dilution of calf thymus DNA was hybridized with a biotinylated DNA probe and then incubated with streptavidin phycoerythrin. After washing, the fluorescence signal was detected on a Packard ScanArray 5000 using three different detection configurations: 488 nm excitation (argon-ion laser)/570 nm emission filter (left); 543.5 nm excitation (He—Ne laser)/570 nm emission filter (middle); 543.5 nm excitation (He—Ne laser)/592 nm emission filter (right).|
Streptavidin Phycoerythrin Conjugate Selection Guide
|Luminex® Assay ||Yes|
|Quantity||1 mL||1 mL||0.5 mL||2 mL|
A phycoerythrin-labeled detection reagent can be used in combination with a green-fluorescent detection reagent to detect two different signals using simultaneous excitation with the 488 nm spectral line of the argon-ion laser (Figure 3). By conjugating R-PE to longer wavelength–emitting fluorescence acceptors, an energy transfer cascade is established wherein excitation of the R-PE produces fluorescence of the acceptor dye by the process of fluorescence resonance energy transfer (FRET).
- Learn More about Fluorescence Resonance Energy Transfer (FRET)—Technical Note 1.2
|Figure 3. Simultaneous detection of three cell surface markers using an Alexa Fluor® 610–R-phycoerythrin tandem streptavidin conjugate, Alexa Fluor® 488 dye and R-phycoerythrin labels. Lymphocytes from ammonium chloride RBC–lysed whole blood were labeled with a biotinylated mouse anti–human CD3 monoclonal antibody washed with 1% BSA in PBS and then incubated with Alexa Fluor® 610–R-phycoerythrin tandem dye–labeled streptavidin. Cells were again washed and then labeled with directly conjugated primary antibodies against the CD8 (Alexa Fluor® 488 labeled) and CD4 (R-phycoerythrin–labeled) markers. After a further wash in 1% BSA/PBS, labeling was analyzed on a Becton Dickinson FACScan flow cytometer using excitation at 488 nm. CD8 was detected in the green channel (525 + 10 nm), CD4 in the orange channel (575 + 10 nm) and CD3 in the red channel (>650 nm). The bivariate scatter plots show the expected mutually exclusive populations of CD4 and CD8 positive cells (panel A), together with co-positive CD3/CD4 (panel B) and CD3/CD8 (panel C) populations.|
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