08 Labeling Chemistry

HiLyte Fluor™ 555 Labeling Kit - NH2

HiLyte Fluor™ 555 Labeling Kit - NH<sub>2</sub>

Antibody / Protein Labeling

  • Product code
    LK14  HiLyte Fluor™ 555 Labeling Kit - NH2
Unit size Price Item Code
3 samples $349.00 LK14-10
3 samples
Quantity may be adjusted within the cart.
3 samples ・NH2-Reactive HiLyte FluorTM 555
・WS Buffer
・Reaction Buffer
・Filtration Tube
3 tubes
4 ml x1
500 μl x1
3 tubes


Product Description
HiLyte Fluor* 555 Labeling Kit-NH2 is used mainly for the preparation of red fluorescence-labeled proteins, such as IgG, for immunostaining, and cellular proteins for tracing. NH2-reactive HiLyteFluor 555, 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 555 can label up to 200 μg of IgG, conjugating about 4 to 6 HiLyte Fluor 555 molecules per IgG molecule. The labeling process is simple-add the NH2-reactive HiLyte Fluor 555 to IgG solution on a membrane and incubate at 37ºC for 10 minutes. The excess HiLyte Fluor 555 molecules can be removed by a filtration tube. The excitation and emission wavelengths of the HiLyte Fluor 555-labeled IgG are 555 nm and 570 nm, respectively (Fig. 2).* HiLyte Fluor is a trademark of AnaSpec, Inc.

Fig. 1 Fluorescence Spectrum of HiLyte Fluor 555-conjugated IgG

Fig. 2 Fluorescence spectrum of HiLyte Fluor 555-conjugated IgG
______excitation spectrum
______emission spectrum

♦ The molecular weight of the protein to be labeled with this kit should be greater than 50,000.
♦ IgG or HiLyte Fluor 555-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 greater than than 10,000, such as BSA or gelatin, purify the IgG solution before labeling HiLyte Fluor 555 with this kit. 
♦ If the IgG solution contains small insoluble materials, centrifuge the solution and use the supernatant for the labeling.

Developer Dojindo Molecular Technologies, Inc.



Open References

1) A. Nagao, K. Sato, K.M. Nishida, H. Siomi, M.C. Siomi, "Gender-specific hierarchy in nuage localization of PIWI-interacting RNA factors in Drosophila"Front Genet.,2011, 2, 55.
2) H. Hoshino, G. Shioi, S. Aizawa, "AVE protein expression and visceral endoderm cell behavior during anterior–posterior axis formation in mouse embryos: Asymmetry in OTX2 and DKK1 expression"Dev. Biol.., 2015, 402, (2), 175.
3) T. Kaneko, T. Minohara, S. Shima, K. Yoshida, A. Fukuda, N. Iwamori, T. Inai and H. Iida, "A membrane protein, TMCO5A, has a close relationship with manchette microtubules in rat spermatids during spermiogenesis"Mol. Reprod. Dev.., 2019, 86, (3), 330.
4) Y. Nakashima, T. Mima, M. Yashiro, T. Sonou, M. Ohya, A. Masumoto, S. Yamanaka, D. Koreeda, K. Tatsuta, Y. Hanba, M. Moribata, S. Negi, T. Shigematsu, "Expression and localization of fibroblast growth factor (FGF) 23 and Klotho in the spleen: its physiological and functional implications"Growth Factors ., 2016, 34, (5-6), 196.
5) Z. Zhang, K. Kakutani, K. Maeno, T. Takada, T. Yurube, M. Doita, M. Kurosaka and K. Nishida, "Expression of silent mating type information regulator 2 homolog 1 and its role in human intervertebral disc cell homeostasis"Arthritis Res. Ther.., 2011, 13, (6), R200.
6) I. Hasan, M. Gerdol, Y. Fujii and Y. Ozeki, "Functional Characterization of OXYL, A SghC1qDC LacNAc-specific Lectin from The Crinoid Feather Star Anneissia Japonica."Mar Drugs., 2019, 17, DOI:10.3390/md17020136. 
7)Y. Fujii, M. Gerdol, S.M.A. Kawsar, I. Hasan, F. Spazzali, T. Yoshida, Y. Ogawa, S. Rajia, K. Kamata, Y. Koide, S. Sugawara, M. Hosono, J.R.H.  Tame, H. Fujita, A. Pallavicini and Y. Ozeki, "A GM1b/asialo-GM1 oligosaccharide-binding R-type lectin from purplish bifurcate mussels Mytilisepta virgata and its effect on MAP kinases."FEBS J., 2019, DOI: 10.1111/febs.15154. 

Q & A


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 555 (prepared at step 3) to


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 555 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.

Handling and storage condition

Handling and storage condition
0-5°C, Protect from moisture

Product Classification

Product Classification

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