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.
Comparison of fluorescence intensity of Phycobiliproteins and cyanine dye
Excitation wavelength: 650 nm
Emission wavelength: 660 nm
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.
Q. Can I use this kit for F(ab)'2?
A. Yes, please follow the labeling protocol for IgG. The recovery of the conjugate should be over 80%.
Q. Can I use this kit for other proteins?
A. Yes, if the molecular weight of the reduced form is greater than 50,000 and it has a reactive SH group, or a disulfide group that can be reduced without losing activity. Follow the labeling protocol for IgG, and use 0.5-1 nmol of sample protein.
Q. How many R-PE molecules per reduced IgG are introduced?
A. The average number of APC molecule per reduced IgG is 1 to 2.
Q. Do I have to use the Filtration tube prior to labeling the protein?
A. If the protein solution does not contain small molecules with reactive SH groups and the concentration of the protein is 10 mg per ml, or about 70 μM, there is no need to use the Filtration tube. Just mix 10 μl of the sample solution with Reaction buffer and add the mixture to a vial of the SH-reactive R-PE.
Q. Do I have to use WS buffer included with the kit?
A. Yes. Use the WS buffer to prepare a stock solution of the conjugate. However, you can choose any kind of buffer appropriate to dilute the conjugate stock solution for your experiment.