Phycoerythrin conjugation

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). 

There are three major classes of conjugated phyco-biliproteins,273/277 all of which are a(3 heterodimers often associated as (a(3)6 (Fig. 23-24). The allophyco-cyanins carry one bilin per subunit, the phycocyanins carry one on the a and two on the (t subunit, and the phycoerythrins carry two or three on the a subunit and three on the P (Fig. 23-24). Cysteine a-84 is one of the frequent attachment sites.273 Three-dimensional structures are known for several of these proteins278 281... 

The effects of CVS on the numbers of T-cell subsets were examined on day 9 at the time of tumor i.v. rechallenging, Fig. (6)-Protocol B. Cells were stained with fluorescein 5-isothiocyanate (FITC)-, phycoerythrin (PE)-conjugated and/or biotinylated mAb and were additionally stained with RED-613-streptavidin for flow-cytometric three-color analysis. 

Small chemical fluorochromes, such as fluorescein, have an advantage in this case because of the stability and predictability of their conjugates. Although methods for calibration of phycoerythrin-labeled antibodies are now available, a wider range of options is available for fluorescein (see Section 3.3.5.). 

Fig. 2. Typical flow cytometry profiles obtained in HT29 cells for (A) total PCNA detection using methanol fixation, (B) Ki-67 expression using methanol fixation, (C) demonstration of tightly bound PCNA using NP-40 extraction, and (D) simultaneous staining of tightly bound PCNA and Ki-67 using Triton X-100 extraction. In D, PCNA was detected using PCIO-FITC, whereas Ki-67 was labeled using a phycoerythrin-conjugated second antibody.

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. 

Photons, 60-62, 66 Phototoxic dyes, 172 Phycoerythrin (PE), 64t, 69t, 252 from algae for conjugation to antibodies, 70-71 in tandem dyes, 71 on calibration beads, 96 providing autofluorescent signatures for flow analysis of algae, 203, 204f, 205f... 

To be of use in microscopy or flow cytometry, this bond needs to be visualized (to the eye or to the photodetector) by the addition of a fluorescent tag. Visualization can be accomplished by one of two different methods. With direct staining, cells are incubated with a monoclonal antibody that has been previously conjugated to a fluorochrome (for example, fluorescein or phycoerythrin or any fluorochrome with appropriate absorption and emission spectra). This procedure is quick and direct it merely involves a half-hour incubation of cells with antibody (at 4°C), followed by several washes to remove weakly or nonspecifically bound antibodies. Cells thus treated are ready for flow analysis (although final fixation with 1% electron microscopic-grade formaldehyde will provide a measure of biological safety and long-term stability). 

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