ICG Labeling Kit - NH2
is used primarily for the preparation of ICG (Iindocyanin green)-labeled antibody for near-infrared fluorescence imaging. ICG offers two remarkable properties:
1) ICG has a strong near-infrared fluorescence even after a few days under physiological conditions. The excitation and emission wavelength of the ICG-labeled proteins are 774 nm and 805 nm, respectively.
2) ICG has been used in clinical fields such as a hepatic deficiency testing. Therefore, ICG and ICG conjugates are materials suitable for in vivo imaging.
This kit contains all required compornents required for labeling, including storage buffer for conjugates. The labeling process is simple:. Add NH2
-reactive ICG to protein solution on a filter membrane, and incubate at 37ºC, for 10 minutes. A filtration tube can remove excess ICG molecules.
Fig. 1 Fluorescein Labeling Process to IgG
♦ If the IgG solution contains other proteins with molecular weight greater than 10,000, such as serum albumin or gelatin, purify the IgG solution before labeling fluorescein with this kit. Commercially available antibody may contain BSA or gelatin as a stabilizer. Dojindo offers IgG Purification Kit-A (AP01-10) and IgG Purification Kit-G (AP02-10) for the purification of the IgG solution
Current Publication Using This Product
Cristian Urla, Guido Seitz, et al., Successful in vivo tumor visualization using fluorescence laparoscopy in a mouse model of disseminated alveolar rhabdomyosarcoma. Surgical Endoscopy. 2014 August
1) M. Ogawa, C. A. S. Regino, J. Seidel, M. V. Green, W. Xi, M. Williams, N. Kosaka, P. L. Choyke and H. Kobayashi, "Dual-Modality Molecular Imaging Using Antibodies Labeled with Activatable Fluorescence and a Radionuclide for Specific and Quantitative Targeted Cancer Detection"
, Bioconjugate Chem
, 20(11), 2177.
2) M. Ogawa, N. Kosaka, P. L. Choyke and H. Kobayashi, "In vivo Molecular Imaging of Cancer with a Quenching Near-Infrared Fluorescent Probe Using Conjuates of Monoclonal Antibodies and Indocyanine Green"
, Cancer Res
, 69(4), 1268.
3) N. Kosaka, M. Ogawa, P. L. Choyke and H. Kobayashi, "Clinical implications of near-infrared fluorescence imaging in cancer"
, Future Oncology
, 5(9), 1501.
4) S. Ito, N.Muguruma, S. Hayashi, S. Taoka, T. Bando, K. Inayama, M. Sogabe, T. Okahisa, S. Okamura, H. Shibata, T. Irimura, K. Takesako and S. Shibamura, "Development of Agents for Reinforcement of Fluorescence on Near-infrared Ray Excitation for Immunohistological Staining"
, Bioorg. Med. Chem
, 6, 613.
5) S. Ito, N. Muguruma, Y. Kakehashi, S. Hayashi, S. Okamura, H. Shibata, T. Okahisa, M. Kanamori, S. Shibamura, K. Takesako, M. Nozawa, K. Ishida and M. Shiga, "Development of Fluorescence-Emitting Antibody Labeling Substance by Near-Infrared Ray Excitation"
, Bioorg. Med. Chem. Lett
, 5, 2689.
6) K. Inayama, S. Ito, N. Muguruma, Y. Kusaka, T. Bando, Y. Tadatsu, M. Tadatsu, K. Ii, S. Shibamura and K. Takesako, "Basic Study of an Agent for Reinforcement of Near-infrared Fluorescence on Tumor Tissue"
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7) S. Ito, N. Muguruma, S. Hayashi, S. Taoka, T. Bando, Y. Kusaka, M. Yano, S. Ichikawa, A. Hiasa, T. Omoya, H. Honda, I. Shimizu, K. Ii, K. Nakamura, K. Takesako, Y. Goto and S. Shibamura, "Visualization of Human Gastric Cancer with a Novel Infrared Fluorescent Labeling Marker of Anti-carcinoembryonic Antigen Antibody in vitro"
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8) S. Taoka, S. Ito, N. Muguruma, S. Hayashi, Y. Kusaka, K. Ii, K. Nakamura, K. Imaizumi, K. Takesako and S. Shibamura, "Reflected Illumination-type Imaging System for the Development of Infrared Fluorescence Endoscopy"
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9) S. Ito, N. Muguruma, S. Hayashi, S. Taoka, A. Tsutsui, T. Fukuda, T. Okahisa, Y. Ohkita, H. Matsunaga, I. Shimizu, K. Nakamura, K. Imaizumi, K. Takesako and S. Shibamura,"Development of an Imaging System Using Fluorescent Labeling Substances Excited by Infrared Rays"
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10) S. Ito, N. Muguruma, Y. Kusaka, M. Tadatsu, K. Inayama, Y. Musashi, M. Yano, T. Bando, H. Honda, I. Shimizu, K. Ii, K. Takesako, H. Takeuchi and S. Shibamura, "Detection of Human Ganstric Cancer of Resected Specimens Using a Novel Infrared Fluorescent Anti-Human Carcinoembryonic Antigen Antibody with an Infrared Fluorescence Endoscope in Vitro"
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Fig. 3 Fluorescent Property of ICG Dye
Q. Can I use this kit for F(ab')2?
A. Yes, please follow the labeling protocol for IgG. The recovery of the conjugate should be over 80%.
Q. Can I use this kit for other proteins or peptides?
A. Yes, if the molecular weight of the reduced form is greater than 50,000 or less than 5,000, and it has a reactive SH group, or a disulfide group that can be reduced without losing activity. If the molecular weight is greater than 50,000, follow the labeling protocol for IgG, and use 0.5-1 nmol of sample protein for LK09-10. If the molecular weight is less than 5,000, follow the labeling protocol for small molecules. If the molecular weight is between 5,000 and 50,000, contact our customer service at email@example.com or 1-877-987-2667 for more information.
Q. Can I use this kit to label oligopeptides or oligonucleotides?
A. Yes, if the molecular weights of the oligonucleotide or the oligopeptide are less than 5,000 and they have at least one SH group. Follow the labeling protocol for small molecule.
Q. What is the minimum amount of IgG that can be labeled with LK13-10?
A. The minimum amount is 50 μg. There is no significant difference in sensitivity and background between 50 μg and 200 μg of IgG. However, even 10 μg IgG can be labeled using 1/5 volume of SH-reactive peroxidase solution at Step 8.
Q. How many alkaline phosphatase molecules per reduced IgG are introduced?
A. The average number of alkaline phosphatase molecule per reduced IgG is 1 to 2.
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 reactive SH groups and the concentration of the protein is 10 mg per ml, or about 70 μM, there is no need to use the Filtration tube. Just mix 10 μl of the sample solution with Solution B and add the mixture to a vial of the SH-reactive peroxidase.
Q. Do I have to use Storage buffer included with the kit?
A. No, you don’t have to use Storage buffer from the kit. You can choose any kind of buffer appropriate for your experiment.
Q. My sample contains small insoluble material. What should I do?
A. Spin the sample and use the supernatant for labeling.
Q. Does unconjugated SH-reactive ALP still have a reactive maleimide after the labeling reaction to IgG?
A. No, Nearly 100% of SH-reactive ALP is used for the IgG
labeling or the small molecule labeling.
Q. Does Storage buffer contain animal products or polymers?
A. No, Storage buffer does not contain any animal products , polymers, or heavy metal ions.