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iFluor® 560 maleimide

Product key features

  • Superior Solubility: Enhanced aqueous solubility for efficient biomolecule conjugation.
  • High Photostability: High quantum yield and stability facilitate the detection of low-abundance targets with greater sensitivity
  • Versatile Conjugation: Maleimide chemistry enables efficient and stable labeling of thiol groups on proteins, antibodies, and oligonucleotide thiophosphates
  • Ex/Em of the conjugate: 560/571 nm
  • Extinction coefficient:  120,000 cm-1M-1
  • Spectrally similar dyes: Cy3, Cy3B

Product description

Cy3 is recognized as the dimmest fluorophore in the Cy dye family. To overcome this limitation, Cy3B was developed as an enhanced derivative, exhibiting a substantial increase in fluorescence quantum yield and photostability. Despite these improvements, Cy3B's limited aqueous solubility presents significant challenges for its efficient conjugation to biomolecules. In response, iFluor® 560 has been introduced, offering enhanced water solubility while retaining spectral properties closely aligned with both Cy3B and Cy3, positioning it as a robust alternative for the development of bioconjugates with bright orange fluorescence. It is optimally excited by 532 nm and 561 nm laser lines and is fully compatible with TRITC filter sets, facilitating its integration into existing experimental workflows. iFluor® 560 is suitable for labeling a wide range of antigens, including cell surface, intracellular, and intranuclear targets. It performs exceptionally well in complex multicolor panels, bridging the gap between fluorophores like FITC and PE. In simpler panels, iFluor® 560 can effectively replace PE to minimize emission spreading into PE tandem dyes. This dye allows for high molar ratio conjugation to proteins with minimal self-quenching, resulting in brighter conjugates. Its robust fluorescence quantum yield and photostability make iFluor® 560 ideal for detecting low-abundance biological targets, delivering greater precision and sensitivity in quantitative fluorescence assays.

The maleimide derivative of iFluor® 560 is widely used for labeling biomolecules with free thiol (SH) groups, including antibodies, proteins, thiol-modified oligonucleotides, and low molecular weight ligands. Maleimides react readily with sulfhydryl groups, forming stable thio-ether bonds between the dye and the biomolecule, facilitating robust and reliable labeling for diverse experimental applications.

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® 560 maleimide Stock Solution (Solution B)
  1. Prepare a 10 mM iFluor® 560 maleimide stock solution by adding anhydrous DMSO to the vial of iFluor® 560 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® 560 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® 560 maleimide per mole of antibody. The following steps outline how to determine the DOS of iFluor® 560 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 = 560 nm for iFluor® 560 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® 560 maleimide, it is at 560 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® 830 maleimide830867----
iFluor® 514 maleimide5115277500010.8310.2650.116
iFluor® 660 maleimide66367825000010.2610.070.08
iFluor® 670 maleimide67168220000010.5510.030.033
iFluor® 560-dUTP *1 mM in TE Buffer (pH 7.5)*56057112000010.5710.04820.069
iFluor® 720 maleimide71674024000010.1410.150.13
Show More (22)

References

View all 43 references: Citation Explorer
The Effect of Conjugated Nitrile Structures as Acceptor Moieties on the Photovoltaic Properties of Dye-Sensitized Solar Cells: DFT and TD-DFT Investigation.
Authors: Tommalieh, Maha J and Aljameel, Abdulaziz I and Hussein, Rageh K and Al-Heuseen, Khalled and Alghamdi, Suzan K and Alrub, Sharif Abu
Journal: International journal of molecular sciences (2024)
Illuminating Neuropeptide Y Y4 Receptor Binding: Fluorescent Cyclic Peptides with Subnanomolar Binding Affinity as Novel Molecular Tools.
Authors: Gleixner, Jakob and Kopanchuk, Sergei and Grätz, Lukas and Tahk, Maris-Johanna and Laasfeld, Tõnis and Veikšina, Santa and Höring, Carina and Gattor, Albert O and Humphrys, Laura J and Müller, Christoph and Archipowa, Nataliya and Köckenberger, Johannes and Heinrich, Markus R and Kutta, Roger Jan and Rinken, Ago and Keller, Max
Journal: ACS pharmacology & translational science (2024): 1142-1168
Fluorescence based HTS-compatible ligand binding assays for dopamine D3 receptors in baculovirus preparations and live cells.
Authors: Tahk, Maris-Johanna and Laasfeld, Tõnis and Meriste, Elo and Brea, Jose and Loza, Maria Isabel and Majellaro, Maria and Contino, Marialessandra and Sotelo, Eddy and Rinken, Ago
Journal: Frontiers in molecular biosciences (2023): 1119157
Characterization of Fluorescent Dyes Frequently Used for Bioimaging: Photophysics and Photocatalytical Reactions with Proteins.
Authors: Archipowa, Nataliya and Wittmann, Lukas and Köckenberger, Johannes and Ertl, Fabian J and Gleixner, Jakob and Keller, Max and Heinrich, Markus R and Kutta, Roger Jan
Journal: The journal of physical chemistry. B (2023): 9532-9542
Possible frequent multiple mitochondrial DNA copies in a single nucleoid in HeLa cells.
Authors: Pavluch, Vojtěch and Špaček, Tomáš and Engstová, Hana and Dlasková, Andrea and Ježek, Petr
Journal: Scientific reports (2023): 5788
Page updated on December 13, 2024

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Catalog Number71684
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Physical properties

Molecular weight

1282.51

Solvent

DMSO

Spectral properties

Correction Factor (260 nm)

0.0482

Correction Factor (280 nm)

0.069

Extinction coefficient (cm -1 M -1)

1200001

Excitation (nm)

560

Emission (nm)

571

Quantum yield

0.571

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 (e.g., iFluor® 560 maleimide) 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 (e.g., iFluor® 560 maleimide) 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 (e.g., iFluor® 560 maleimide) 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.