Phycobiliproteins derivatives

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). 

Conjugates of (strept)avidin with these fluorescent probes may be prepared by activation of the phycobiliprotein with N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) to create a sulf-hydryl-reactive derivative, followed by modification of (strept)avidin with 2-iminothiolane or SATA (Chapter 1, Section 4.1) to create the free sulfhydryl groups necessary for conjugation. The protocol for SATA modification of (strept)avidin can be found in Section 3.1, this chapter. The procedure for SPDP activation of phycobiliproteins can be found in Chapter 9, Section 7. Reacting the SPDP-activated phycobiliprotein with thiol-labeled (strept)avidin at a molar ratio of 2 1 will result in highly fluorescent biotin binding probes. 

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. 

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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