08 Labeling Chemistry

HiLyte Fluor™ 647 Labeling Kit - NH2

HiLyte Fluor™ 647 Labeling Kit - NH<sub>2</sub>

Antibody / Protein Labeling

  • Only 1 hour to recover conjugates
  • All processes in a single filtration tube
  • High recovery of conjugates
  • Applicable for 50-200 μg IgG
  • Product code
    LK15  HiLyte Fluor™ 647 Labeling Kit - NH2
Unit size Price Item Code
3 samples $317.00 LK15-10
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3 samples
Quantity may be adjusted within the cart.
Component
3 samples ・NH2-Reactive HiLyte FluorTM 647
・WS Buffer
・Reaction Buffer
・Filtration Tu
3 tubes
4 ml×1
500 μl×1
3 tubes

Description

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
Blue line: excitation spectrum
Purple line: emission spectrum

Precaution
♦ 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. 
♦ If the IgG solution contains small insoluble materials, centrifuge the solution and use the supernatant for the labeling.

Developer Dojindo Molecular Technologies, Inc.

Manual

References

Open References

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.
4) T. Kojima, J. Hata, H. Oka, K. Hayashi, K. Hitomi and H. Nakano, "Spatial arrangement of proteins using scCro-tag: application for an in situ enzymatic microbead assay."Biosci. Biotechnol. Biochem., 2018, 82, (11), 1911.
5) S. Mawaribuchi, Y. Haramoto, H. Tateno, Y. Onuma, Y. Aiki and Y. Ito, "rBC2LCN lectin as a potential probe of early-stage HER2-positive breast carcinoma."FEBS Open Bio., 2020, DOI: 10.1002/2211-5463.12852.

Q & A

Q

Can I use this kit for other proteins?

A

Yes, if the molecular weight is greater than 50,000.

Q

Do I have to use a Filtration tube prior to labeling the protein?

A

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.

Q

How long is the HiLyte Fluor 555-labeled protein stable?

A

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.

Q

What is the minimum amount of IgG that can be labeled by this kit?

A

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.

Q

Can I use this kit to label oligonucleotides or peptides?

A

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
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