Phycobiliproteins are fluorescent proteins derived from cyanobacteria and eukaryotic algae. Their fluorescence is much higher than 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-NH2 is for simple and rapid preparation of APC-labeled IgG (Fig. 2). NH2-reactive APC (a component of this kit) has an activated ester group and can easily make a covalent bond with an amino group of the target molecule without any activation process. The filtration tube in this kit is used for buffer exchange and concentration of sample IgG solution. This kit contains all the reagents necessary for APC labeling including the storage buffer for conjugates.
Fig. 1 Fluorescence spectrum of APC
Excitation wavelength: 650 nm
Emission wavelength: 660 nm
Fig. 2 IgG labeling reaction of NH2-reactive APC
♦ The molecular weight of the 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 greater 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.
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