AAT Bioquest

ICG-Osu *UltraPure Grade*

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Physical properties
Molecular weight828.03
Spectral properties
Correction Factor (280 nm)0.076
Extinction coefficient (cm -1 M -1)230000
Excitation (nm)789
Emission (nm)813
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
Alternative formats


Molecular weight
Correction Factor (280 nm)
Extinction coefficient (cm -1 M -1)
Excitation (nm)
Emission (nm)
Indocyanine green (ICG) is a cyanine dye used in medical diagnostics for determining cardiac output, hepatic function, and liver blood flow, as well as ophthalmic angiography. With a peak spectral absorption near 800 nm, ICG operates in the infrared spectrum, penetrating retinal layers effectively. This property allows ICG angiography to capture deeper circulation patterns compared to fluorescein angiography. ICG tightly binds to plasma proteins, remaining confined to the vascular system. It has a half-life of 150 to 180 seconds and is exclusively removed by the liver, directed to bile juice. Recent research highlights ICG's ability to target atheromas within 20 minutes of injection, facilitating the in vivo detection of lipid-rich, inflamed, coronary-sized plaques in atherosclerotic rabbits. In experiments using ex vivo fluorescence reflectance imaging, rabbits injected with ICG demonstrated higher plaque target-to-background ratios compared to those injected with saline. An amino-reactive ICG derivative plays a crucial role in forming ICG bioconjugates with antibodies and other biological molecules. This ultra-pure grade ICG-Osu has a minimum purity exceeding 97% and moderate water solubility.

Example protocol


Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

Protein stock solution (Solution A)
  1. Mix 100 µL of a reaction buffer (e.g., 1 M  sodium carbonate solution or 1 M phosphate buffer with pH ~9.0) with 900 µL of the target protein solution (e.g., antibody, protein concentration >2 mg/mL if possible) to give 1 mL protein labeling stock solution.

    Note: The pH of the protein solution (Solution A) should be 8.5 ± 0.5. If the pH of the protein solution is lower than 8.0, adjust the pH to the range of 8.0-9.0 using 1 M  sodium bicarbonate solution or 1 M pH 9.0 phosphate buffer.

    Note: The protein should be dissolved in 1X phosphate-buffered saline (PBS), pH 7.2-7.4. If the protein is dissolved in Tris or glycine buffer, it must be dialyzed against 1X PBS, pH 7.2-7.4, to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for protein precipitation.

    Note: Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well. The presence of sodium azide or thimerosal might also interfere with the conjugation reaction. Sodium azide or thimerosal can be removed by dialysis or spin column for optimal labeling results.

    Note: The conjugation efficiency is significantly reduced if the protein concentration is less than 2 mg/mL. For optimal labeling efficiency, the final protein concentration range of 2-10 mg/mL is recommended.

ICG-OSu stock solution (Solution B)
  1. Add anhydrous DMSO into the vial of ICG-OSu to make a 10 mM stock solution. Mix well by pipetting or vortex.

    Note: Prepare the dye stock solution (Solution B) before starting the conjugation. Use promptly. Extended storage of the dye stock solution may reduce the dye activity. Solution B can be stored in the freezer for two weeks when kept from light and moisture. Avoid freeze-thaw cycles.


This labeling protocol was developed for the conjugate of Goat anti-mouse IgG with ICG-OSu. You might need further optimization for your particular proteins.

Note: Each protein requires a distinct dye/protein ratio, which also depends on the properties of dyes. Over-labeling of a protein could detrimentally affect its binding affinity while the protein conjugates of low dye/protein ratio give reduced sensitivity.

Run conjugation reaction
  1. Use a 10:1 molar ratio of Solution B (dye)/Solution A (protein) as the starting point:  Add 5 µL of the dye stock solution (Solution B, assuming the dye stock solution is 10 mM) into the vial of the protein solution (95 µL of Solution A) with effective shaking. The concentration of the protein is ~0.05 mM assuming the protein concentration is 10 mg/mL and the molecular weight of the protein is ~200KD.

    Note: We recommend using a 10:1 molar ratio of Solution B (dye)/Solution A (protein). If it is too low or too high, determine the optimal dye/protein ratio at 5:1, 15:1, and 20:1, respectively.

  2. Continue to rotate or shake the reaction mixture at room temperature for 30-60 minutes.
Purify the conjugation

The following protocol is an example of dye-protein conjugate purification by using a Sephadex G-25 column.

  1. Prepare the Sephadex G-25 column according to the manufacturer's instructions.

  2. Load the reaction mixture (From "Run conjugation reaction") to the top of the Sephadex G-25 column.

  3. Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top resin surface.

  4. Add more PBS (pH 7.2-7.4) to the desired sample to complete the column purification. Combine the fractions that contain the desired dye-protein conjugate.

    Note: For immediate use, the dye-protein conjugate need be diluted with staining buffer, and aliquoted for multiple uses.

    Note: For longer-term storage, the dye-protein conjugate solution needs to be concentrated or freeze-dried.


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of ICG-Osu *UltraPure Grade* to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM120.769 µL603.843 µL1.208 mL6.038 mL12.077 mL
5 mM24.154 µL120.769 µL241.537 µL1.208 mL2.415 mL
10 mM12.077 µL60.384 µL120.769 µL603.843 µL1.208 mL

Molarity calculator

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Spectral properties

Correction Factor (280 nm)0.076
Extinction coefficient (cm -1 M -1)230000
Excitation (nm)789
Emission (nm)813

Product Family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Correction Factor (280 nm)
ICG-ATT [3-ICG-acyl-1,3-thiazolidine-2-thione]7898132300000.076
ICG amine7898132300000.076
ICG acid7898132300000.076
ICG Maleimide7898132300000.076
ICG azide7898132300000.076
ICG alkyne7898132300000.076
ICG hydrazide7898132300000.076