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IR820 N-succinimidyl ester

Content and storage

Material Amount Storage Stability
IR820-NHS 1 mg
  • -20 °C
  • Desiccate
  • Protect from light
When stored as directed, reactive probes are stable for at least 3 months
10 mg
50 mg
100 mg

General Specifications

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

Formula: C58H61N3O11S2

Shipping Condition: Blue Ice

Regulatory Statement: For Research Use Only. Not for use in diagnostic procedures.

Introduction

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.

Guidelines for use

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.

Experimental Protocols

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.

For example:

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.

References

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

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