Fluorescein phycobiliproteins

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). 

Preparation of a series of phycobiliprotein tandem dyes allows multiplexed analysis of different targets in a sample. In addition, since RPE can be excited by the argon-ion laser at 488 nm, a fluorescein-labeled probe can be used concurrently with RPE alone and RPE-tandem conjugates to create a multiplexed system of different fluorescent probes that can be used simultaneously. Table 9.3 shows the different combinations of dyes that can be used in this type of assay with RPE and APC. 

Antibodies labeled with fluorescent molecules have several applications, particularly in cytochemistry and cell sorting. There are many fluorochromes used in labeling (1), such as coumarin derivatives, phycobiliproteins, and rare earth chelates however, fluorescein and rhodamine (Table 1) are the most commonly used. 

Heterogeneous Fluorophore Fluorescein Europium chelate Phycobiliproteins Fluorescence intensity Time-resolved fluorescence Fluorescence intensity... 

The use of single laser excitation for multiparameter fluorescence measurements is more challenging. What is needed is a series of fluorophores that all excite at the same laser wavelength, typically the 488 nm argon ion laser line, but which emit at different distinguishable wavelengths. One series of such labels includes fluorescein, R-PE, and either Cy5-labeled R-PE or Texas Red-labeled R-PE tandem conjugates or another phycobiliprotein, PerCP. Fluorescein emits at 525 nm, R-PE at 575 nm, and Cy5-PE at 670 nm, and each can be detected with minimum spillover from the others. ... 

The term probe is normally used as a synonym of label. Thus, in contrast to labels, probes respond to their microenvironment or to a chemical species. Those probes responding to a chemical species such as oxygen, an ion, or to pH are also referred to as indicators. In this article, both terms will be used indistinctly. Different classes of fluorescent labels are available. Low molecular weight dyes include xan-thene (rhodamines, fluoresceins), cyanine, coumar-ins, sulfonated pyrenes, and metal phthalocyanine compounds, while high molecular weight labels include phycobiliproteins and other luminescent proteins. 

Commonly used probes are fluorescein derivatives, rhodamine derivatives, polycyclic aromatic hydrocarbons, coumarines, amine reagents such as fluoresca-mine or NBD-Cl, phycobiliproteins, porphyrins, and metal chelates. The latter may show fluorescence,... 

There are many fluorophores used in antibody labelling such as rhodamine, coumarin derivatives, phycobiliproteins, and rare earth chelates, however fluorescein is probably the most widely used. Individual choice may be determined by the optical filters available for the microscope or other detector. Protocol 3 describes the use of fluorescein-N-hyroxysuccinimide ester to label IgG amino groups (3). Fluorescein isothiocyanate may also be used for the same purpose but it requires harsher conditions. The reaction is quenched by the addition of ethanolamine and excess labelling reagent is removed by gel filtration. 

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