Phycobiliproteins are fluorescent proteins derived from cyanobacteria and eukaryotic algae. Their fluorescence is much higher than that of chemical fluorescent probes such as fluorescein and rhodamine. Allophycocyanin (APC) is one of the phycobiliproteins, and it has a red fluorescence at around 660 nm (Fig. 1). Because of this high fluorescence, phycobiliprotein-labeled antibodies and other molecules can provide greater sensitivity in flow cytometry and immunostaining. Allophycocyanin Labeling Kit-SH is for simple and rapid preparation of APC-labeled IgG (Fig. 2). SH-reactive APC (a component of this kit) has a maleimide group and can easily make a covalent bond with a sulfhydryl group of the target molecule without any activation process. The filtration tube in this kit allows a quick buffer exchange and concentration of sample IgG solution. This kit contains all the reagents necessary for APC labeling, including a reducing agent for preparation of reduced IgG that has an SH group and a storage buffer for conjugates.
Fig. 1 Comparison of fluorescence intensity of Phycobiliproteins and cyanine dye
Excitation wavelength: 650 nm
Emission wavelength: 660 nm
Fig. 2 IgG labeling reaction of SH-reactive APC
♦ The molecular weight of the reduced protein to be labeled with this kit should be greater than 50,000.
♦ IgG or allophycocyanin-conjugated IgG is always on the membrane of the filtration tube during the labeling process.
♦ If the IgG solution contains other proteins with molecular weight larger than 10,000, such as BSA or gelatin, purify the IgG solution before labeling allophycocyanin with this kit. IgG solution can be purified by IgG Purification Kits (not included in this kit).
♦ If the IgG solution contains small insoluble materials, centrifuge the solution and use the supernatant for the labeling.