||When stored as directed, reactive probes are stable for at least 3 months|
Color: Dark brown powder Label: Succinimidyl ester Product Size: 1 kit
Detection Method: Fluorescence, optoacoustic Excitation Class: Near infrared, NIR Excitation/Emission (nm): 710/820
Molecular Weight: 1040.26 g.mol-1
Shipping Condition: Blue Ice
Regulatory Statement: For Research Use Only. Not for use in diagnostic procedures.
IR820 is a non-invasive near-infrared (NIR) fluorescence imaging dye that belongs to the family of the heptamethine dyes and has a rigid cyclohexenyl ring, increasing molecular stability and photostability. IR820 could be used as both an imaging dye and a hyperthermia agent.1
IR820 has a little absorption in the visible range thus exhibit low autofluorescence, tissue absorbance, and scatter at NIR wavelengths (700-900 nm).
The succinimidyl esters (NHS) of the IR820 dye offer the opportunity to develop optimal conjugates. Succinimidyl ester active groups provide an efficient and convenient way to selectively link IR820 dyes to primary amines (R-NH2) on various substrates (Antibodies, peptides, proteins, nucleic-acid, small molecule drugs etc.). Succinimidyl esters have very low reactivity with aromatic amines, alcohols, and phenols, including tyrosine and histidine.
Immediately before use, dissolve the IR820-NHS dyes in anhydrous dimethylformamide (DMF). Once reconstituted, this reactive probe solution is somewhat unstable, especially in presence of moisture that can slowly hydrolyze the succinimidyl ester to the non-reactive carboxylic acid.
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.
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.
1. Fernandez-Fernandez A., Manchanda R., Lei T., Carvajal D.A., Tang Y., Kazmi S.Z.R., and McGoron A.J., Comparative Study of the Optical and Heat Generation Properties of IR820 and Indocyanine Green, Molecular Imaging, 2012, 11, 99–113