Fluorescent Proteins
Phycoerythrins
B-Phycoerythrin (B-PE) and the closely related R-Phycoerythrin (R-PE) are the most intensely fluorescent phycobiliproteins having orange fluorescence. They are significantly brighter and more photostable than conventional organic fluorophores. Both are composed of α, β and γ subunits and are present as (αβ)6γ.
B-PE was isolated from cyanobacteria and eukaryotic algae. Its primary absorption peak is at 545 nm with a secondary peak at 563 nm. B-PE labeled streptavidin, primary and secondary antibodies have been widely used in applications such as flow cytometry and multi-color immunofluorescent staining.
R-PE was isolated from red algae. Its primary absorption peak is at 565 nm with secondary peaks at 496 and 545 nm. The broad excitation spectrum provides the advantage for multi-color immunofluorescent staining or cell sorting.
SMCC activated B-PE or R-PE are generated by chemically modifying the proteins. SMCC reacts with the primary amine on B-PE or R-PE and introduces maleimide groups. These maleimide groups easily react with thiol groups of target proteins without the need for any additional activation, resulting in convenient conjugation of B-PE or R-PE with proteins.
Phycocyanins
Allophycocyanin (APC), a highly fluorescent phycobiliprotein, is made up of α and β subunits and is present as a trimer (αβ)3, which is unstable and susceptible to dissociation at low concentrations. The monomer αβ has a lower fluorescence quantum yield compared to the trimer and the maximal absorption is shifted to 620 nm. The chemically cross-linked APC trimer (CL-APC) is much more stable than the native APC trimer, while retaining the same spectroscopic properties. APC labeled streptavidin, primary and secondary antibodies have been widely used in applications such as flow cytometry, live cell staining, and immunofluorescent staining.
In SMCC-modified CL-APC, the SMCC group reacts with the primary amine on CL-APC and introduces maleimide groups to APC. These maleimide groups easily react with thiol groups of target proteins without the need for any additional activation, resulting in convenient conjugation of APC with proteins.
C-Phycocyanin (C-PC) represents the major phycobiliprotein in many cyanobacteria and is the second phycobiliprotein in some red algae. The pigment has a single visible absorption maximum between 615 and 620 nm and a fluorescence emission maximum at ~650 nm. Its molecular mass is between 70.000 and 110.000 Da. The pigment is composed of two subunits, α and β, which occur in equal numbers, but the exact number of α and β pairs which make up the molecule may vary among the species. Both α and β subunits contain only the PCB chromophore. In addition to absorbing light directly, this intensely blue pigment accepts quanta from phycoerythrin by fluorescent energy transfer in organisms in which PE is present. The red fluorescence of C-PC is transferred to allophycocyanin.