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iFluor® 810 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 emission 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 emerging developments aim to achieve high-resolution, multimodality, and lifetime-based in vivo fluorescence imaging. Our iFluor® 810 is designed to label proteins and other biomolecules with infrared fluorescence. Conjugates prepared with iFluor® 810 have excitation and emission in the IR range. iFluor® 810 dye emission 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 detections. iFluor® 810 maleimide is thiol-reactive and can be readily used to conjugate thiol-containing biomolecules.

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® 810 maleimide Stock Solution (Solution B)
  1. Prepare a 10 mM iFluor® 810 maleimide stock solution by adding anhydrous DMSO to the vial of iFluor® 810 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® 810 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 the 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.

  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® 810 maleimide per mole of antibody. The following steps outline how to determine the DOS of iFluor® 810 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 = 822 nm for iFluor® 810 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® 810 maleimide, it is at 822 nm. To ensure accurate readings, make sure the conjugate is free of any nonconjugated dye.

Calculate DOS

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

https://www.aatbio.com/tools/degree-of-labeling-calculator

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of iFluor® 810 maleimide to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM65.55 µL327.751 µL655.501 µL3.278 mL6.555 mL
5 mM13.11 µL65.55 µL131.1 µL655.501 µL1.311 mL
10 mM6.555 µL32.775 µL65.55 µL327.751 µL655.501 µL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
/=x=

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® 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® 830 maleimide830867----
iFluor® 514 maleimide5115277500010.8310.2650.116
iFluor® 660 maleimide66367825000010.2610.070.08
iFluor® 670 maleimide67168220000010.5510.030.033
iFluor® 720 maleimide71674024000010.1410.150.13
Show More (20)

Citations

View all 3 citations: Citation Explorer
Tumor cytotoxicity and immunogenicity of a novel V-jet neon plasma source compared to the kInpen
Authors: Miebach, Lea and Freund, Eric and Horn, Stefan and Niessner, Felix and Sagwal, Sanjeev Kumar and von Woedtke, Thomas and Emmert, Steffen and Weltmann, Klaus-Dieter and Clemen, Ramona and Schmidt, Anke and others,
Journal: Scientific Reports (2021): 1-14
Tumor cytotoxicity and immunogenicity of a novel V-jet neon plasma source compared to the kINPen
Authors: Miebach, Lea and Freund, Eric and Horn, Stefan and Niessner, Felix and Sagwal, Sanjeev Kumar and von Woedtke, Thomas and Emmert, Steffen and Weltmann, Klaus-Dieter and Clemen, Ramona and Schmidt, Anke and others,
Journal: Scientific Reports (2021): 1--14
Nanovesicle delivery to the liver via retinol binding protein and platelet-derived growth factor receptors: how targeting ligands affect biodistribution
Authors: Hsu, Ching-Yun and Chen, Chun-Han and Aljuffali, Ibrahim A and Dai, You-Shan and Fang, Jia-You
Journal: Nanomedicine (2017)

References

View all 18 references: Citation Explorer
A target cell-specific activatable fluorescence probe for in vivo molecular imaging of cancer based on a self-quenched avidin-rhodamine conjugate
Authors: Hama Y, Urano Y, Koyama Y, Kamiya M, Bernardo M, Paik RS, Shin IS, Paik CH, Choyke PL, Kobayashi H.
Journal: Cancer Res (2007): 2791
Fluorescence imaging in vivo: recent advances
Authors: Rao J, Dragulescu-Andrasi A, Yao H.
Journal: Curr Opin Biotechnol (2007): 17
Ex vivo fluorescence imaging of normal and malignant urothelial cells to enhance early diagnosis
Authors: Steenkeste K, Lecart S, Deniset A, Pernot P, Eschwege P, Ferlicot S, Leveque-Fort S, Bri and et R, Fontaine-Aupart MP.
Journal: Photochem Photobiol (2007): 1157
In vivo monitoring the fate of Cy5.5-Tat labeled T lymphocytes by quantitative near-infrared fluorescence imaging during acute brain inflammation in a rat model of experimental autoimmune encephalomyelitis
Authors: Berger C, Gremlich HU, Schmidt P, Cannet C, Kneuer R, Hiest and P, Rausch M, Rudin M.
Journal: J Immunol Methods (2007): 65
A protocol for imaging alternative splicing regulation in vivo using fluorescence reporters in transgenic mice
Authors: Bonano VI, Oltean S, Garcia-Blanco MA.
Journal: Nat Protoc (2007): 2166
Page updated on October 8, 2024

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Catalog Number1388
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Physical properties

Molecular weight

1525.55

Solvent

DMSO

Spectral properties

Correction Factor (260 nm)

0.09

Correction Factor (280 nm)

0.15

Extinction coefficient (cm -1 M -1)

2500001

Excitation (nm)

811

Emission (nm)

822

Quantum yield

0.051

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
UNSPSC12171501
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.
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.
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.