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ICG Labeling Kit - NH2

Item # Unit Size
1 sample (labeling 100 ug)
3 samples (labeling 100 ug)

For Research Use Only Products

  • Suitable wavelength for In Vivo imaging
  • Quick and Easy Labeling to Antibodies : 1.5hr
  • High Recovery Rate : more than 90%
Contents of the Kit
LK31-08: 1 sample
NH2-reactive ICG  :1 tube  Reaction buffer  :250 μl x 1 tube 
WS buffer  :1.5 ml x 1 bottle   Filtration tube  :1 tube 

LK31-10: 3 samples
NH2-reactive ICG  :3 tubes  Reaction buffer  :500 μl x 1 tube 
WS buffer  :4 ml x 1 bottle   Filtration tube  :3 tubes

Storage: 0-5ºC
Shipping Condition: ambient temperature

Product Description
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 conjugatesare 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. N. Muguruma, et al., Antibodies Labeled with Fluorescence-agent Excitable by Infrared Rays. J Gastroenterol. 1988;33:467-471.
2. S. Ito, et al., Development of Fluorescence-Emitting Antibody Labeling Substance by Near-Infrared Ray Excitation. Bioorg Med Chem Lett. 1995;5:2689-2694.
3. S. Ito, et al., Development of an Imaging System Using Fluorescent Labeling Substances Excited by Infrared Rays. Dig Endoscopy. 1997;9:278-282.
4. S. Ito, et al., Development of Agents for Reinforcement of Fluorescence on Near-infrared Ray Excitation for Immunohistological Staining. Bioorg Med Chem. 1998;6:613-618.
5. S. Taoka, et al., Reflected Illumination-type Imaging System for the Development of Infrared Fluorescence Endoscopy. Dig Endoscopy. 1999;11:321-326.
6. N. Muguruma, et al., Labeled Carcinoembryonic Antigen Antibodies Excitable by Infrared Rays: a Novel Diagnostic Method for Micro Cancers in the Digestive Tract. Intern Med. 1999;38:537-542.
7. S. Ito, et al., Visualization of Human Gastric Cancer with a Novel Infrared Fluorescent Labeling Marker of Anti-carcinoembryonic Antigen Antibody in vitro. Dig Endoscopy. 2000;12:33-36.
8. S. Ito, et al., 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. Endoscopy. 2001;33:849-853.
9. T. Bando, et al., Basic Study on a Labeled anti-mucin Antibody Detectable by Infrared-fluorescence Endoscopy. J Gastroenterol. 2002;37:260-269.
10. K. Inayama, et al., Basic Study of an Agent for Reinforcement of Near-infrared Fluorescence on Tumor Tissue. Dig Liver Dis. 2003;35:88-93.
11. M. Ogawa, et al., Dual-Modality Molecular Imaging Using Antibodies Labeled with Activatable Fluorescence and a Radionuclide for Specific and Quantitative Targeted Cancer Detection. Bioconjug Chem. 2009;20:2177-2184.
12. M. Ogawa, et al., In vivo Molecular Imaging of Cancer with a Quenching Near-Infrared Fluorescent Probe Using Conjuates of Monoclonal Antibodies and Indocyanine Green. Cancer Res. 2009;69:1268-1272.
13. N. Kosaka, et al., Clinical implications of near-infrared fluorescence imaging in cancer. Future Oncol. 2009;5:1501-1511.

Fig. 3 Fluorescent Property of ICG Dye

Can I use this kit for F(ab')2?
Yes, please follow the labeling protocol for IgG. The recovery of the conjugate should be over 80%.

Can I use this kit for other proteins or peptides?
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 info@dojindo.com or 1-877-987-2667 for more information.

Can I use this kit to label oligopeptides or oligonucleotides?
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.

What is the minimum amount of IgG that can be labeled with LK13-10?
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.

How many alkaline phosphatase molecules per reduced IgG are introduced?
The average number of alkaline phosphatase molecule per reduced IgG is 1 to 2.

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

Do I have to use Storage buffer included with the kit?
No, you don’t have to use Storage buffer from the kit. You can choose any kind of buffer appropriate for your experiment.

My sample contains small insoluble material. What should I do?
Spin the sample and use the supernatant for labeling.

Does unconjugated SH-reactive ALP still have a reactive maleimide after the labeling reaction to IgG? No. Nearly 100% of SH-reactive ALP is used for the IgG
labeling or the small molecule labeling.

Does Storage buffer contain animal products or polymers?
No, Storage buffer does not contain any animal products , polymers, or heavy metal ions.