The ICG-NHS dye can virtually be conjugated to any primary amine-containing molecule such as peptides, proteins, antibodies, small-molecule drugs… If possible avoid nucleophilic bases, since it will partially degrades ICG core structure to non-NIR-fluorescent by-products.
Example of conjugation:
- Antibody conjugation: Typical antibody conjugation reactions are carried out in 0.1 M sodium bicarbonate buffer, pH 8.5, at room temperature for 2 hour and protected from light. We recommend trying different molar ratio between antibodies and reactive dyes in order to reach your needs. Labeled antibodies are typically separated from free ICG dye using a gel filtration column, such as SephadexTM G-25, BioGel® P-30, or equivalent. For much larger or smaller proteins, select a gel filtration media with an appropriate molecular weight cut-off or purify by dialysis. Keep labeled antibody at 4°C in PBS. The number of fluorochrome per antibody will vary depending on the molar ratio between antibodies and reactive dyes. An average number of fluorochrome per antibody can be determined by spectrophotometric analysis or/and by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry.
Antibody (250 ug) is diluted in 0.1 M NaHCO3 (pH 8.5) to 2 mg/mL (125 uL) (other buffer could be used such as Na2HPO4 0.1M, pH 8.5). 4 to 8 equivalents of fluorescent ICG-NHS dye (from stock solution in DMSO or DMF at 1 mM) is added to the antibody solution and incubated for 0.5 to 2 hours. After incubation, antibody can be purified by centrifuge filtration using desalting Zeba spin column 7K (0.5 mL) and store in PBS at 4°C. The number of fluorochromes per antibody was determined by spectrophotometric analysis and determined to be approximately 2, 3 ICG dyes per antibody using respectively 4 and 8 molar equivalent of ICG-NHS.
For ICG/Ab calculation, take UV absorbance at 280nm and 800nm and apply following calculation:
A800: Absorbance at 800nm
A280: Absorbance at 280nm
ε: Molar extinction coefficient of protein (for Antibody ~ 203000 L.mol-1.cm-1)
22884: molar extinction coefficient of ICG in PBS at 800 nm
0.052: Correction factor for ICG
- Small molecule conjugation: Typical small-molecule conjugation reactions are carried out in dry dimethylformamide (DMF) or dimethylsulfoxide (DMSO) with an organic base such diisopropylethylamine (DIPEA) at room temperature for 1-3 hours and protected from light. Labeled molecules are typically purified by reverse-phase HPLC and appropriate fractions are lyophilized.