HiLyte Fluor* 647 Labeling Kit-NH2 is mainly used for the preparation of red fluorescence-labeled proteins, such as IgG, for immunostaining, and cellular proteins for tracing. NH2-reactive HiLyteFluor 647, a component of this kit, has a succinimidyl group (NHS) that reacts with amino groups on proteins or other molecules (Fig. 1). This kit contains all the reagents necessary for labeling. Each tube of HiLyte Fluor 647 can label up to 200 μg of IgG, conjugating about 4 to 6 HiLyte Fluor 647 molecules per IgG molecule. The labeling process is simple-add the NH2-reactive HiLyte Fluor 647 to IgG solution on a membrane and incubate at 37ºC for 10 minutes. The excess HiLyte Fluor 647 molecules can be removed by a filtration tube. The excitation and emission wavelengths of the HiLyte Fluor 647-labeled IgG are 652 nm and 673 nm, respectively (Fig. 2). *HiLyte Fluor is a trademark of AnaSpec, Inc.
Fig. 1 Fluorescence Spectrum of HiLyte Fluor 647-conjugated IgG
Fig. 2 Fluorescence spectrum of HiLyte Fluor 647-conjugated IgG
♦ The molecular weight of the protein to be labeled with this kit should be greater than 50,000.
♦ IgG or HiLyte Fluor 647-conjugated IgG is always on the membrane of the filtration tube during the labeling process.
♦ If the IgG solution contains other proteins with molecular weights larger than 10,000, such as BSA or gelatin, purify the IgG solution before labeling HiLyte Fluor 647 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. S. Hiroyasu, T. Ozawa, H. Kobayashi, M. Ishii, Y. Aoyama, Y. Kitajima, T. Hashimoto, J.C.R. Jones and D. Tsuruta, “Bullous pemphigoid IgG induces BP180 internalization via a macropinocytic pathway”, Am. J. Pathol.., 2013, 182, (3), 828.
2. W. Jin, K. Yamada, M. Ikami, N. Kaji, M. Tokeshi, Y. Atsumi, M. Mizutani, A. Murai, A. Okamoto, T. Namikawa, Y. Baba, M. Ohta, “Application of IgY to sandwich enzyme-linked immunosorbent assays, lateral flow devices, and immunopillar chips for detecting staphylococcal enterotoxins in milk and dairy products”, J. Microbiol. Methods., 2013, 92, (3), 323.
3. Y. Hayashi, M. Okutani, S. Ogawa, T. Tsukahara, R. Inoue, “Generation of anti-porcine CD69 monoclonal antibodies and their usefulness to evaluate early activation of cellular immunity by flow cytometric analysis”, Anim. Sci. J.., 2018, 89, (5), 825.