The IR820-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 IR820 core structure to non-NIR-fluorescent by-products.
Example of conjugation:
1. 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 IR820 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.
A solution of Antibody (2 mg/mL, 125 uL) in 0.1 M NaHCO3 (pH 8.5) was incubated with 4, 8 or 20 equivalents of fluorescent IR820-NHS dye for 2 hours. After incubation, the antibody was purified by centrifuge filtration using 30000 Dalton molecular weight cutoff filters and wash two times with PBS. Finally, labeled antibody was purified by gel filtration using Zeba spin column and store in PBS at 4°C. The number of fluorochromes per antibody was determined by spectrophotometric analysis and determined to be approximately 2, 4 and 9 IR820 dye per antibody using respectively 4, 8 and 20 molar equivalent of IR820-NHS.
2. 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.