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Protocol |
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Before use, centrifuge the R-PE suspension at 10,000g for 10 min at 4°C. Discard the supernatant and resuspend the pellet (R-PE) into the desired buffer. Desalt the sample using either Sephadex G-25 or dialysis. Store R-PE at 4°C and keep away from light.
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Substrate/Buffer |
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R-PE is supplied in potassium phosphate buffer, pH 7.0 with ammonium sulfate. The protein is very stable and can be stored for years in this buffer.
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References |
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1. Glazer, AN. and L. Stryer, Methods Enzymol. 184, 188 (1990).
2. Oi, VT. et al. J. Cell Biol. 93, 981 (1982).
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Notes |
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R-PE (R-Phycoerythrin),1 a fluorescent protein, belongs to the phycobiliprotein family of highly soluble and fluorescent proteins derived from cyanobacteria and eukaryotic algae. R-PE is made of α, β and γ subunits and is present as (αβ)6γ. The polymer is very stable since it does not dissociate even when diluted to 10-12 M.
The protein has broad absorption bands with peaks at 565 nm (εM =1.96 X 106 M-1cm-1), 498 (εM =1.53 X 106 M-1cm-1), and 539 nm (εM =1.62 X 106 M-1cm-1); consequently, it can be excited with versatile excitation sources. The broad excitation spectrum provides the advantage for multi-color immunofluorescent staining or cell sorting. For example, a sample labeled with fluorescein and R-PE can be excited with a single light source at 488 nm but detected at 520 nm and 575 nm, respectively. R-PE and the closely related B-PE are the most intensely fluorescent phycobiliproteins. They are significantly brighter and more photostable than conventional organic fluorophores.2
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