R-phycoerythrin

There are three main classes of phycobiliproteins, differing in their protein structure, bilin content, and fluorescent properties. These are phycoerythrin, phycocyanin, and allo-phycocyanin (APC). There are two main forms of phycoerythrin proteins commonly in use B-phycoerythrin isolated from Porphyridium cruentum and R-phycoerythrin from Gastroclonium coulteri. There also are three main forms of pigments found in these proteins phycoerythrobilin, phycourobilin, and phycocya no bilin (Glazer, 1985). The relative content of these pigments in the phycobiliproteins determines their spectral properties. All of them,... 

The spectral properties of four major phycobiliproteins used as fluorescent labels can be found in Tables 9.1 and 9.2. The bilin content of these proteins ranges from a low of four prosthetic groups in C-phycocyanin to the 34 groups of B- and R-phycoerythrin. Phycoerythrin derivatives, therefore, can be used to create the most intensely fluorescent probes possible using these proteins. The fluorescent yield of the most luminescent phycobiliprotein molecule is equivalent to about 30 fluoresceins or 100 rhodamine molecules. Streptavidin-phycoerythrin conjugates, for example, have been used to detect as little as 100 biotinylated antibodies bound to receptor proteins per cell (Zola et al., 1990). 

Fig. 2.146. Outline of full process of extraction, isolation and purification of R-phycoerythrin from G. verrucosa. Reprinted with permission from G. Wang [319].

Fluorescence detection relies on the visualization of a secondary antibody that has been labeled with a fluorophore such as fluorescein (FITC), Texas Red, Tetramethyl rhodamine (TRITC), or R-phycoerythrin. Although this method of detection has a reduced sensitivity of twofold to fourfold compared to chemiluminescence detection, it presents a tenfold greater linear dynamic range, thus providing better linearity and better quantiflcation within the detection limits. Since secondary antibodies can be labeled with fluor-ophores of distinct colors, multiplexing (simultaneous detection of several antigens) of the same blot is feasible. 

Abbreviations AMCA, 7-amino-4-methylcoumarin B-PE, B phycoerythrin Cy, cyanine DAMC, diethylaminocoumarin FITC, fluorescein isothiocyanate RB-200-SC, lissamine rhodamine sulfonylchloride R-PE, R phycoerythrin SITS, 4-acetamido-4 -isothiocyanato-stilbene-2,2 -disulfonic acid TRITC, tetramethyl rhodamine isothiocyanate XRITC, rhodamine X isothiocyanate. Information obtained from refs. 2, 9, and 10. 

Most use of phycobilliproteins has involved B and R phycoerythrins, which yield the highest fluorescence intensities. They are also the largest phyco-... 

Fig. 5.2. Methods for detection of passenger-ligand interaction. (A) Fluorophore-coupled ligand (B) fluorophore-coupled antibody (C) quaternary complex generated by subsequent rounds of incubation with ligand, primary antibody, biotinylated second antibody, and strep ta-vidin, R-phycoerythrin conjugate (D) quaternary complex generated by subsequent rounds of incubation wi tli ligand, primary antibody, biotinylated second antibody, and streptavidin-coated magnetobeads. L ligand P passenger.

Streptavidin, R-phycoerythrin conjugate, lmgmL-1 (Molecular Probes). 

The target molecule can be directly labeled with a fluorescent dye or it can be indirectly labeled via biotinylation and successive incubation with a streptaviclin, R-phycoerythrin conjugate. We recommend to biotinylate the target protein since it can be used both for MACS and for FACS then. For direct fluorescence labeling of the target molecule, NHS-coupled fluorescence dyes, e. g., Alexa Fluor 488 from Molecular Probes, can be used, following the procedure described in protocol 2. 

If the ligand protein is fluorescently labeled, resuspend the cells in 10 pL PBS and subject directly to FACS. If the ligand protein is biotinylated (protocol 2), resuspend the cells in 10 pL PBS containing streptavidin, R-phycoerythrin conjugate (1 10 dilution in PBS). Incubate on ice again for 10 min. 

Argon ion 488 Fluorescein, R-phycoerythrin, PerCP, PE-tandems, EGFP, EYFP, propidium iodide, Alexa 488, acridine orange... 

Fluorescence Emission Spectra of Fluorescein and R-Phycoerythrin (Excitation at 488 nm)... 

Handelman et al. (1999) treated milled oat groat pearlings, trichomes, flour, and bran with methanol and tested the fractions for antioxidant capacity against low-density lipoprotein (LDL) oxidation and R-phycoerythrin protein oxidation in the oxygen radical absorbance capacity... 

ORAC R-Phycoerythrin ABAP Fluorimetric (area under curve) C6, C15... 

TRAP with 15 nM R-phycoerythrin, 4 mM Fluorescence (495 nm/ 0.96% Blood plasma (heparin... 

Urine 5.73 0.58 mmol/24 hr (elderly women) ORAC with R-phycoerythrin (Cl 5) C13... 

Coelho, L., Prince, J., and Nolen, T.G., Processing of defensive pigment in Aplysia califomica acquisition, modification and mobilization of the red algal pigment r-phycoerythrin by the digestive gland, J. Exp. Biol., 201, 425, 1998. 

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