Contents of the Kit
|NH2-reactive HiLyte Fluor 647
||:4 ml x 1 bottle
||:0.5 ml x 1 tube
Storage Condition: 0-5ºC
Shipping Condition: ambient temperature
Required Equipment and Materials
Microcentrifuge, 10 μl and 100-200 μl adjustable pipettes, 37ºC incubator, 0.5 ml microtubes, DMSO
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.
Can I use this kit for other proteins?
Yes, if the molecular weight is greater than 50,000.
Do I have to use a Filtration tube prior to labeling the protein?
If the protein solution does not contain small molecules with an amino group and the concentration of the protein is 10 mg per ml, or about 70 μM, there is no need to use the Filtration tube. Mix 10 μl of the sample solution with 90 μl of Reaction buffer and add 8 μl NH2-reactive HiLyte Fluor 647 (prepared at step 3) to the mixture, and follow the protocol starting at step 4.
How long is the HiLyte Fluor 555-labeled protein stable?
If you store at 4oC, it is stable for over 2 months. For longer storage, add 100% volume of glycerol, aliquot, and store at -20oC. However, please note that stability depends on the protein itself.
What is the minimum amount of IgG that can be labeled by this kit?
The minumum amount of IgG is 10 μg. For less than 20 μg, follow the manual and add 4 μl NH2-reactive HiLyte Fluor 647 instead of 8 μl at step 4.
Can I use this kit to label oligonucleotides or peptides?
No. Oligonucleotides and peptides may be too small to retain on the membrane filter of the Filtration tube.