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iFluor® IRD 800 CW Maleimide

Product key features

  • The same chemical structure as the IRDye® 800 CW Maleimide
  • A NIR Dye for labeling antibodies
  • Multiple applications, e.g., In-Cell Western, in vivo imaging

Product description

iFluor® IRD 800 CW Maleimide has the same chemical structure as the IRDye® 800 CW Maleimide (IRDye® is the trademark of LI-COR). IRD 800CW Maleimide is a thiol-reactive near-infrared (NIR) fluorescent dye with good water solubility. It can be used to label peptides, antibodies, and other thiol-containing biomolecules such as thiol-modified oligos. The IRD 800CW conjugates can be used as a probe for Western blot, flow cytometry, immunocytochemical assays, including In-Cell Western assays and On-Cell Western cell-based assays, protein arrays, microscopy, tissue section imaging, and in vivo imaging. IRD 800CW dye-conjugated probes are also used for fluorescence imaging-based cancer diagnosis.

Example protocol

PREPARATION OF STOCK SOLUTIONS

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

Prepare iFluor® IRD 800 CW Maleimide stock solution
  1. Allow the vial of iFluor® Dye maleimide to warm up to room temperature.
  2. Add anhydrous DMSO to the vial to prepare a 10 mM dye stock solution.
  3. Vortex the vial briefly to fully dissolve the dye, and then centrifuge to collect the dye at the bottom of the vial.
  4. Protect all stock solutions from light as much as possible by wrapping containers in aluminum foil.
Prepare antibody or protein solution for labeling
  1. If your protein already contains a thiol group, prepare the protein at 50-100 uM (for example: 5mg/ml BSA is ~75uM) in 50~100 mM MES buffer or buffers of your choice with pH 6.5~7.0.
  2. If labeling with an intact antibody, reduction of disulfide bonds need to be carried out before maleimide reaction. Prepare antibody in 2-10 mg/ml in a suitable buffer with pH 7.0–7.5. A 10-fold molar excess of a reducing agent such as DTT or TCEP is added to the antibody. If DTT is used, it must be removed by dialysis or desalting to a suitable buffer with pH 6.5~7.0 prior to conjugation. If TCEP is used, it is not necessary to remove excess TCEP during conjugation with maleimides, however, removal of TCEP by dialysis or desalting prior to conjugation gives the better labeling efficiency.

    Below is a sample protocol for generating free thiol groups on antibody:
    1. Prepare 2-10mg/ml IgG solution in PBS.
    2. Prepare a fresh solution of 1 M DTT (15.4 mg/100 µL) in distilled water. 
    3. Add 1- 20 µL of DTT stock per ml of IgG solution while mixing. 
    4. Let the solution stand at room temperature for 30 minutes without additional mixing (to minimize the re-oxidation of cysteines to cystines). 
    5. Pass the reduced IgG over a filtration column pre-equilibrated with 50 mM MES buffer (pH=6.5) to remove excess DTT.
    6. Determine the antibody concentrations. This can be done either spectrophotometrically or colorimetrically.
    7. Carry out the conjugation as soon as possible after this step.

    Note: For the best results, IgG solutions should be > 2 mg/mL.

    Note: The reduction can be carried out in almost any buffer from pH 7 to 7.5, e.g., MES, phosphate, or TRIS buffers.

    Note: Steps 5 can be replaced by dialysis.

     
  3. If your protein doesn’t have a free thiol group or disulfide bond to reduce, a thiolation modification need to be carried out before maleimide conjugation (for example:  using 2-Iminothiolane or 2-IT) to introduce sulfhydryl (-SH) groups to the original amino groups on protein.

SAMPLE EXPERIMENTAL PROTOCOL

This labeling protocol was developed for the labeling IgG with iFluor® Dye maleimide. Further optimization may be required for your specific 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 10~20:1 molar ratio of iFluor® Dye maleimide : IgG as the starting point. While stirring or vortexing the protein solution, add a volume of dye stock solution to result in a dye: protein molar ratio of 10-20. For example, for 5mg/ml IgG (~33 uM), you would add dye to a final concentration of 0.33-0.66 mM.

    Note: We recommend using a 10:1 molar ratio of dye to protein.  If the ratio 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. Purify the conjugate on a gel filtration column, such as a Sephadex G-25 column or equivalent matrix, or by extensive dialysis at 4°C in an appropriate buffer.

Recommended AAT Desalting Columns:

Volume of Reaction Catalog#
0.6-1.0mL

Cat#60504: PD-10 Column

https://www.aatbio.com/products/readiuse-disposable-pd-10-desalting-column?unit=60504
~0.1mL

Cat#60500: Spin Column

https://www.aatbio.com/products/readiuse-bio-gel-p-6-spin-column?unit=60500 

Optional: Characterize the Desired Dye-Protein Conjugate

Determining the Degree of Substitution (DOS) is crucial in characterizing dye-labeled proteins. Lower DOS proteins tend to have weaker fluorescence, but higher DOS proteins may also have reduced fluorescence. For most antibodies, the optimal DOS is between 2 and 10, depending on the dye and protein properties. For effective labeling, the degree of substitution should be controlled to have 5-8 moles of iFluor® IRD 800 CW Maleimide to one mole of antibody. The following steps are used to determine the DOS of iFluor® IRD 800 CW Maleimide-labeled proteins:

  1. Measure absorption— To measure the absorption spectrum of a dye-protein conjugate, the sample concentration should be kept between 1 and 10 µM (For example: IgG conjugate: 10uM is ~1.5mg/ml), depending on the dye's extinction coefficient. 
  2. Read OD (absorbance) at 280 nm and dye maximum absorption (ƛ max = 624 nm for iFluor® IRD 800 CW Maleimide dyes). For most spectrophotometers, the sample (from the column fractions) must be diluted with de-ionized water so that the OD values range from 0.1 to 0.9. The O.D. (absorbance) at 280 nm is the maximum absorption of protein, while 774 nm is the maximum absorption of iFluor® IRD 800 CW Maleimide. To obtain accurate DOS, ensure the conjugate is free of the non-conjugated dye.
  3. Calculate DOS using our DOS calculator: https://www.aatbio.com/tools/degree-of-labeling-calculator

Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
iFluor® IRD 800 CW SE77479224000010.090.30.3

References

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Bionanocomposite MIL-100(Fe)/Cellulose as a high-performance adsorbent for the adsorption of methylene blue.
Authors: Abbasi, Shahla and Nezafat, Zahra and Javanshir, Shahrzad and Aghabarari, Behzad
Journal: Scientific reports (2024): 14497
Experimental upper bounds for resonance-enhanced entangled two-photon absorption cross section of indocyanine green.
Authors: He, Manni and Hickam, Bryce P and Harper, Nathan and Cushing, Scott K
Journal: The Journal of chemical physics (2024)
Eco-friendly biosynthesis of TiO2 nanoparticles using Desmostachya bipinnata extract: Larvicidal and pupicidal potential against Aedes aegypti and Spodoptera litura and acute toxicity in non-target organisms.
Authors: Shyam-Sundar, Narayanan and Karthi, Sengodan and Senthil-Nathan, Sengottayan and Narayanan, Kilapavoor Raman and Santoshkumar, Balasubramanian and Sivanesh, Haridoss and Chanthini, Kanagaraj Muthu-Pandian and Stanley-Raja, Vethamonickam and Ramasubramanian, Ramakrishnan and Abdel-Megeed, Ahmed and Malafaia, Guilherme
Journal: The Science of the total environment (2023): 159512
Development of ratiometric fluorescent probes based on peptides for sensing Pb2+ in aquatic environments and human serum.
Authors: Lee, Jae Yoon and Mehta, Pramod Kumar and Subedi, Sumita and Lee, Keun-Hyeung
Journal: Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy (2023): 122502
Pyrolyzed magnetic NiO/carbon-derived nanocomposite from a hierarchical nickel-based metal-organic framework with ultrahigh adsorption capacity.
Authors: Beigi, Negar and Shayesteh, Hadi and Javanshir, Shahrzad and Hosseinzadeh, Majid
Journal: Environmental research (2023): 116146
Page updated on May 19, 2025

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1 mg
5 mg
Catalog Number
7161271613
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Physical properties

Molecular weight

1239.33

Solvent

DMSO

Spectral properties

Correction Factor (260 nm)

0.3

Correction Factor (280 nm)

0.3

Extinction coefficient (cm -1 M -1)

2400001

Excitation (nm)

774

Emission (nm)

792

Quantum yield

0.09

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
Product Image
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Gallery Image 1
Fluorescent dye maleimides are the most popular tool for conjugating dyes to a peptide, protein, antibody, thiol-modified oligonucleotide, or nucleic acid through their SH group. Maleimides react readily with the thiol group of proteins, thiol-modified oligonucleotides, and other thiol-containing molecules under neutral conditions. The resulting dye conjugates are quite stable.