Phycobiliprotein Derivatives

The spectral properties of four major phycobiliproteins used as fluorescent labels can be found in Table 3. The bilin content of these proteins ranges from a low of 4 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 rhoda-mine 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). 

Source Porphyridiutn cruentum Gastroclonium coulteri Anabaena variabilis Anabaena variabilis 

Dialyze the phycobiliprotein into 50 mM sodium borate, 0.3 M NaCl, pH 8.5 (note commercial preparations of these proteins come as an ammonium sulfate suspension). After dialysis, adjust the protein solution to a concentration of 1 mg/ml. Higher protein concentrations may be used, but the amount of cross-linking reagent added to each milliliter of the reaction should be proportionally scaled up, as well. Protect the protein solution from undue exposure to light. 

Add 25 xl of the stock solution of either SPDP or LC-SPDP in DMSO to each milliliter of the protein solution. If Sulfo-LC-SPDP is used, add 50 xl of the stock solution in water to each milliliter of protein solution. 

Mix and react for at least 30 min at room temperature. Longer reaction times, even overnight, will not adversely affect the modification. 

Lucifer Yellow CH is soluble in aqueous solution, and it should be stable for awhile if protected from light. The reagent is available as three different salts of the sulfonate groups. The ammonium salt of the fluorophore is soluble to a level of 9 percent in water, while the lithium and potassium salts have a solubility of 5 and 1 percent, respectively. A concentrated stock solution of the fluorophore may be prepared in water and an aliquot added to a buffered reaction medium to facilitate the transfer of small quantities. For aqueous reactions, a pH range of 5-9 will result in efficient hydrazone formation with aldehyde or ketone residues. 

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, 

Source Porphyridium cruentum Gastroclonium coulter i Anabaena variabilis Anabaena variabilis 

Phycobiliprotein Molecular weight Absorption maximum (nm) EC (cm-1 M 1) Emission maximum (nm) Fluorescence QY 

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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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