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iFluor® 830 maleimide

In vivo fluorescence imaging uses a sensitive camera to detect the fluorescence emission from fluorophores in whole-body living small animals. To overcome the photon attenuation in living tissue, fluorophores with long emissions at the infrared (IR) region are generally preferred. Recent advances in imaging strategies and reporter techniques for in vivo fluorescence imaging include novel approaches to improve the specificity and affinity of the probes and to modulate and amplify the signal at target sites for enhanced sensitivity. Further developments aim to achieve high-resolution, multimodality, and lifetime-based in vivo fluorescence imaging. Our iFluor® 830 is designed to label proteins and other biomolecules with infrared fluorescence. Conjugates prepared with iFluor® 830 have excitation and emission maxima in the IR range. iFluor® 830 dye emission is a unique color for spectrum flow cytometry as it is well separated from commonly used far-red fluorophores such as Cy5, Cy7, or allophycocyanin (APC), facilitating multicolor analysis. This fluorophore is also useful for small animal in vivo imaging applications or other imaging applications requiring IR detection. iFluor® 830 maleimide is one of the most convenient reactive forms for preparing the desired iFluor® 830 conjugates. It specifically and readily reacts with thiol groups, such as reduced antibodies and thiol-modified oligos.

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

iFluor® 830 maleimide Stock Solution (Solution B)
  1. Prepare a 10 mM iFluor® 830 maleimide stock solution by adding anhydrous DMSO to the vial of iFluor® 830 maleimide. Mix well by pipetting or vortexing.

    Note: Before starting the conjugation process, prepare the dye stock solution (Solution B) and use it promptly. Prolonged storage of Solution B may reduce its activity. If necessary, Solution B can be stored in the freezer for up to 4 weeks, provided it is protected from light and moisture. Avoid freeze/thaw cycles.

Protein Stock Solution (Solution A)
  1. Prepare a 1 mL protein labeling stock solution, by mixing 100 µL of a reaction buffer (e.g., 100 mM MES buffer with a pH ~6.0) with 900 µL of the target protein solution (e.g., an antibody or protein solution with a concentration >2 mg/mL if possible).

    Note: The pH of the protein solution (Solution A) should be 6.5 ± 0.5.

    Note: Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or other proteins will not be labeled well.

    Note: The conjugation efficiency is significantly reduced if the protein concentration is less than 2 mg/mL. To achieve optimal labeling efficiency, it is recommended to maintain a final protein concentration within the range of 2-10 mg/mL.

Disulfide Reduction (If Necessary)

If your protein does not contain a free cysteine, it must be treated with DTT or TCEP to generate a thiol group. DTT and TCEP are utilized to convert disulfide bonds into two free thiol groups. If using DTT, ensure to remove any free DTT via dialysis or gel filtration before conjugating a dye maleimide to your protein. Below is a sample protocol for generating a free thiol group:

  1. To prepare a fresh solution of 1 M DTT, dissolve 15.4 mg of DTT in 100 µL of distilled water.

  2. To prepare the IgG solution in 20 mM DTT, first, add 20 µL of DTT stock to each milliliter of the IgG solution while mixing gently. Then, allow the solution to stand at room temperature for 30 minutes without additional mixing. This resting period helps to minimize the reoxidation of cysteines to cystines.

  3. Pass the reduced IgG through a filtration column that has been pre-equilibrated with "Exchange Buffer." Collect 0.25 mL fractions as they elute from the column.

  4. Determine the protein concentrations and combine the fractions containing the highest amounts of IgG. This can be accomplished using either spectrophotometric or colorimetric methods.

  5. Proceed with the conjugation immediately after this step (refer to the Sample Experiment Protocol for details).

    Note: IgG solutions should be >4 mg/mL for the best results. The antibody should be concentrated if less than 2 mg/mL. Include an extra 10% for losses on the buffer exchange column.

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

    Note: Steps 3 and 4 can be replaced by dialysis.

SAMPLE EXPERIMENTAL PROTOCOL

This labeling protocol was designed for the conjugation of goat anti-mouse IgG with iFluor® 830 maleimide. You may need to further optimize the protocol for your specific proteins.

Note: Each protein requires a specific dye-to-protein ratio, which varies based on the properties of the dyes. Over-labeling a protein can negatively impact its binding affinity while using a low dye-to-protein ratio can result in reduced sensitivity.

Run Conjugation Reaction
  1. Use a 10:1 molar ratio of Solution B (dye)/Solution A (protein) as the starting point. Add 5 µL of the dye stock solution (Solution B, assuming the dye stock solution is 10 mM) to the vial of the protein solution (95 µL of Solution A), and mix thoroughly by shaking. The protein solution has a concentration of ~0.05 mM assuming the protein concentration is 10 mg/mL and the molecular weight of the protein is ~200KD.

    Note: We recommend using a 10:1 molar ratio of Solution B (dye) to Solution A (protein). If this ratio is not suitable, determine the optimal dye/protein ratio by testing 5:1, 15:1, and 20:1 ratios.

  2. Continue to rotate or shake the reaction mixture at room temperature for 30-60 minutes.

Purify Conjugate

The following protocol serves as an example for purifying dye-protein conjugates using a Sephadex G-25 column.

  1. Follow the manufacturer's instructions to prepare the Sephadex G-25 Column.

  2. Load the reaction mixture (from the "Run conjugation reaction" step) onto the top of the Sephadex G-25 column.

  3. Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top of the resin surface.

  4. Add more PBS (pH 7.2-7.4) to the desired sample to complete the column purification. Then, combine the fractions that contain the desired dye-protein conjugate.

    Note: For immediate use, dilute the dye-protein conjugate with staining buffer. If you need to use it multiple times, divide it into aliquots.

    Note: For long-term storage, the dye-protein conjugate solution should be either concentrated or freeze-dried.

Characterize the Desired Dye-Protein Conjugate

The Degree of Substitution (DOS) is a key factor in characterizing dye-labeled proteins. Proteins with a lower DOS generally have weaker fluorescence intensity, while those with a higher DOS may also have reduced fluorescence. For most antibodies, the optimal DOS is recommended to be between 2 and 10, depending on the properties of the dye and protein. For effective labeling, the DOS should be controlled to have 5-8 moles of iFluor® 830 maleimide per mole of antibody. The following steps outline how to determine the DOS of iFluor® 830 maleimide-labeled proteins.

Measure Absorption

To measure the absorption spectrum of a dye-protein conjugate, maintain the sample concentration between 1 and 10 µM. The exact concentration within this range will depend on the dye's extinction coefficient.

Read OD (absorbance) at 280 nm and dye maximum absorption (ƛmax = 830 nm for iFluor® 830 dyes)

For most spectrophotometers, dilute the sample (from the column fractions) with de-ionized water until the OD values fall within the range of 0.1 to 0.9. The optimal absorbance for protein is at 280 nm, while for iFluor® 830 maleimide, it is at 830 nm. To ensure accurate readings, make sure the conjugate is free of any non-conjugated dye.

Calculate DOS

You can calculate DOS using our tool by following this link:

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® 350 maleimide3454502000010.9510.830.23
iFluor® 488 maleimide4915167500010.910.210.11
iFluor® 555 maleimide55757010000010.6410.230.14
iFluor® 647 maleimide65667025000010.2510.030.03
iFluor® 680 maleimide68470122000010.2310.0970.094
iFluor® 700 maleimide69071322000010.2310.090.04
iFluor® 750 maleimide75777927500010.1210.0440.039
iFluor® 790 maleimide78781225000010.1310.10.09
iFluor® 800 maleimide80182025000010.1110.030.08
iFluor® 810 maleimide81182225000010.0510.090.15
iFluor® 820 maleimide82285025000010.110.16
iFluor® 860 maleimide85387825000010.10.14
iFluor® 532 maleimide5375609000010.6810.260.16
iFluor® 594 maleimide58760320000010.5310.050.04
iFluor® 405 maleimide4034273700010.9110.480.77
iFluor® 430 maleimide4334984000010.7810.680.3
iFluor® 568 maleimide56858710000010.5710.340.15
iFluor® 633 maleimide64065425000010.2910.0620.044
iFluor® 450 maleimide4515024000010.8210.450.27
iFluor® 460 maleimide468493800001~0.810.980.46
iFluor® 665 maleimide667692110,00010.2210.120.09
iFluor® 546 maleimide54155710000010.6710.250.15
iFluor® 840 maleimide8368792000001-0.20.09
iFluor® 770 maleimide77779725000010.160.090.08
iFluor® 780 maleimide78480825000010.1610.130.12
iFluor® 514 maleimide5115277500010.8310.2650.116
iFluor® 660 maleimide66367825000010.2610.070.08
iFluor® 670 maleimide67168220000010.5510.030.033
iFluor® 720 maleimide71674024000010.1410.150.13
iFluor® 560 maleimide56057112000010.5710.04820.069
Show More (21)

References

View all 50 references: Citation Explorer
A bone-targeting drug delivery vehicle of a metal-organic framework conjugate with zoledronate combined with photothermal therapy for tumor inhibition in cancer bone metastasis.
Authors: Ge, Ting and Weiwei, Zhang and Ge, Fei and Zhu, Longbao and Song, Ping and Li, Wanzheng and Gui, Lin and Dong, Wan and Tao, Yugui and Yang, Kai
Journal: Biomaterials science (2022): 1831-1843
Synthesis of sialic acid conjugates of the clinical near-infrared dye as next-generation theranostics for cancer phototherapy.
Authors: Dong, Huiling and Gao, Yanan and Huang, Xuefei and Wu, Xuanjun
Journal: Journal of materials chemistry. B (2022): 927-934
6-Aminocaproic acid as a linker to improve near-infrared fluorescence imaging and photothermal cancer therapy of PEGylated indocyanine green.
Authors: Hu, Qiang and Wang, Kesi and Qiu, Liyan
Journal: Colloids and surfaces. B, Biointerfaces (2021): 111372
Diagnostic imaging in near-infrared photoimmunotherapy using a commercially available camera for indocyanine green.
Authors: Inagaki, Fuyuki F and Fujimura, Daiki and Furusawa, Aki and Okada, Ryuhei and Wakiyama, Hiroaki and Kato, Takuya and Choyke, Peter L and Kobayashi, Hisataka
Journal: Cancer science (2021): 1326-1330
Hepatic bile acid transport increases in the postprandial state: A functional 11C-CSar PET/CT study in healthy humans.
Authors: Ørntoft, Nikolaj W and Gormsen, Lars C and Keiding, Susanne and Munk, Ole L and Ott, Peter and Sørensen, Michael
Journal: JHEP reports : innovation in hepatology (2021): 100288
Page updated on December 1, 2024

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Physical properties

Molecular weight

1603.65

Solvent

DMSO

Spectral properties

Absorbance (nm)

830

Excitation (nm)

830

Emission (nm)

867

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
iFluor® 830 is designed to label proteins and other biomolecules with infrared fluorescence. Conjugates prepared with iFluor® 830 have excitation and emission maxima in the IR range. iFluor® 830 maleimide is one of the most convenient reactive forms for preparing the desired iFluor® 830 conjugates as it specifically and readily reacts with thiol groups such as reduced antibodies and thiol-modified oligos.
iFluor® 830 is designed to label proteins and other biomolecules with infrared fluorescence. Conjugates prepared with iFluor® 830 have excitation and emission maxima in the IR range. iFluor® 830 maleimide is one of the most convenient reactive forms for preparing the desired iFluor® 830 conjugates as it specifically and readily reacts with thiol groups such as reduced antibodies and thiol-modified oligos.
iFluor® 830 is designed to label proteins and other biomolecules with infrared fluorescence. Conjugates prepared with iFluor® 830 have excitation and emission maxima in the IR range. iFluor® 830 maleimide is one of the most convenient reactive forms for preparing the desired iFluor® 830 conjugates as it specifically and readily reacts with thiol groups such as reduced antibodies and thiol-modified oligos.