Phycobiliproteins are fluorescent proteins derived from cyanobacteria and eukaryotic algae. Their fluorescence is much higher than chemical fluorescent probes such as fluorescein and rhodamine. R-phycoerythrin (R-PE) is one of the phycobiliproteins and has an orange fluorescence at around 578 nm, and it can be excited at 488 nm (Fig. 1). Because of this high fluorescence, phycobiliprotein-labeled antibodies or other molecules can give greater sensitivity in flow cytometry and immunostaining. R-phycoerythrin Labeling Kit-SH is for simple and rapid preparation of R-PE-labeled IgG (Fig. 2). SH-reactive R-PE (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 R-PE labeling, including the reducing agent for preparation of reduced IgG that has an SH group and the storage buffer for conjugates.
Fig. 1 Fluorescence spectrum of R-PE
Excitation wavelength: 566 nm
Emission wavelength: 578 nm
Fig. 2 IgG labeling reaction of SH-reactive R-PE
♦ The molecular weight of the reduced protein to be labeled with this kit should be greater than 50,000.
♦ IgG or R-Phycoerythrin-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 prior to labeling R-phycoerythrin 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.
1) M. Hikida, S. Casola, N. Takahashi, T. Kaji, T. Takemori, K. Rajewsky and T. Kurosaki, “PLC-γ2 is essential for formation and maintenance of memory B cells”, J. Exp. Med.., 2009, 206, (3), 681.
2) M. Segawa, S. Fukada, Y. Yamamoto, H. Yahagi, M. Kanematsu, M. Sato, T. Ito, A. Uezumi, S. Hayashi, Y. Miyagoe-Suzuki, S. Takeda, K. Tsujikawa and H. Yamamoto, “Suppression of macrophage functions impairs skeletal muscle regeneration with severe fibrosis”, Exp. Cell Res.., 2008, 314, (17), 3232.
3) M. Yasunaga, S. Saijou, S. Hanaoka, T. Anzai, R. Tsumura, Y. Matsumura, “Significant antitumor effect of an antibody against TMEM180, a new colorectal cancer‐specific molecule”, Cancer Sci.., 2019, 110, (2), 761.