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iFluor® 560 succinimidyl ester

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: Succinimidyl ester chemistry enables efficient and stable labeling of proteins, antibodies, and other amine-containing molecules.
  • Ex/Em of the conjugate: 560/571 nm
  • Extinction coefficient:  120,000 cm-1M-1
  • Spectrally similar dyes: Cy3, Cy3B
  • Molecular weight: 1257.45

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 succinimidyl ester of iFluor® 560 is a widely used reagent for the conjugation of this dye to proteins or antibodies. Succinimidyl esters react selectively and efficiently with primary amines (such as the side chains of lysine residues or aminosilane-coated surfaces) at pH 7-9, forming stable covalent amide bonds. This property makes iFluor® 560 succinimidyl ester an excellent choice for labeling proteins, amine-modified oligonucleotides, and other amine-containing molecules.

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.

1. Protein stock solution (Solution A)
Mix 100 µL of a reaction buffer (e.g., 1 M  sodium carbonate solution or 1 M phosphate buffer with pH ~9.0) with 900 µL of the target protein solution (e.g. antibody, protein concentration >2 mg/mL if possible) to give 1 mL protein labeling stock solution.
Note     The pH of the protein solution (Solution A) should be 8.5 ± 0.5. If the pH of the protein solution is lower than 8.0, adjust the pH to the range of 8.0-9.0 using 1 M  sodium bicarbonate solution or 1 M pH 9.0 phosphate buffer.
Note     The protein should be dissolved in 1X phosphate buffered saline (PBS), pH 7.2-7.4. If the protein is dissolved in Tris or glycine buffer, it must be dialyzed against 1X PBS, pH 7.2-7.4, to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for protein precipitation.
Note     Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well. The presence of sodium azide or thimerosal might also interfere with the conjugation reaction. Sodium azide or thimerosal can be removed by dialysis or spin column for optimal labeling results.
Note     The conjugation efficiency is significantly reduced if the protein concentration is less than 2 mg/mL. For optimal labeling efficiency the final protein concentration range of 2-10 mg/mL is recommended.


2. iFluor™ 560 SE stock solution (Solution B)
Add anhydrous DMSO into the vial of iFluor™ 560 SE to make a 10 mM stock solution. Mix well by pipetting or vortex.
Note     Prepare the dye stock solution (Solution B) before starting the conjugation. Use promptly. Extended storage of the dye stock solution may reduce the dye activity. Solution B can be stored in freezer for two weeks when kept from light and moisture. Avoid freeze-thaw cycles.

SAMPLE EXPERIMENTAL PROTOCOL

This labeling protocol was developed for the conjugate of Goat anti-mouse IgG with iFluor™ 560 SE. You might need further optimization for your particular proteins.
Note     Each protein requires distinct dye/protein ratio, which also depends on the properties of dyes. Over labeling of a protein could detrimentally affects its binding affinity while the protein conjugates of low dye/protein ratio gives reduced sensitivity.


Run conjugation reaction
  1. Use 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) into the vial of the protein solution (95 µL of Solution A) with effective shaking. The concentration of the protein is ~0.05 mM assuming the protein concentration is 10 mg/mL and the molecular weight of the protein is ~200KD.
    Note     We recommend to use 10:1 molar ratio of Solution B (dye)/Solution A (protein). If it is too less 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. Prepare Sephadex G-25 column according to the manufacture instruction.
  2. Load the reaction mixture (From "Run conjugation reaction") to 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 resin surface.
  4. Add more PBS (pH 7.2-7.4) to the desired sample to complete the column purification. Combine the fractions that contain the desired dye-protein conjugate.
    Note     For immediate use, the dye-protein conjugate need be diluted with staining buffer, and aliquoted for multiple uses.
    Note     For longer term storage, dye-protein conjugate solution need be concentrated or freeze dried. 

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of iFluor® 560 succinimidyl ester 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 mM79.526 µL397.63 µL795.26 µL3.976 mL7.953 mL
5 mM15.905 µL79.526 µL159.052 µL795.26 µL1.591 mL
10 mM7.953 µL39.763 µL79.526 µL397.63 µL795.26 µL

Molarity calculator

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

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
iFluor® 350 succinimidyl ester3454502000010.9510.830.23
iFluor® 405 succinimidyl ester4034273700010.9110.480.77
iFluor® 488 succinimidyl ester4915167500010.910.210.11
iFluor® 514 succinimidyl ester5115277500010.8310.2650.116
iFluor® 532 succinimidyl ester5375609000010.6810.260.16
iFluor® 555 succinimidyl ester55757010000010.6410.230.14
iFluor® 594 succinimidyl ester58760320000010.5310.050.04
iFluor® 633 succinimidyl ester64065425000010.2910.0620.044
iFluor® 647 succinimidyl ester65667025000010.2510.030.03
iFluor® 660 succinimidyl ester66367825000010.2610.070.08
iFluor® 680 succinimidyl ester68470122000010.2310.0970.094
iFluor® 700 succinimidyl ester69071322000010.2310.090.04
iFluor® 750 succinimidyl ester75777927500010.1210.0440.039
iFluor® 610 succinimidyl ester61062811000010.8510.320.49
iFluor® 710 succinimidyl ester71673915000010.6010.120.07
iFluor® 790 succinimidyl ester78781225000010.1310.10.09
iFluor® 800 succinimidyl ester80182025000010.1110.030.08
iFluor® 810 succinimidyl ester81182225000010.0510.090.15
iFluor® 820 succinimidyl ester82285025000010.110.16
iFluor® 860 succinimidyl ester85387825000010.10.14
iFluor® 546 succinimidyl ester54155710000010.6710.250.15
iFluor® 568 succinimidyl ester56858710000010.5710.340.15
iFluor® 430 succinimidyl ester4334984000010.7810.680.3
iFluor® 450 succinimidyl ester4515024000010.8210.450.27
iFluor® 840 succinimidyl ester8368792000001-0.20.09
iFluor® 670 succinimidyl ester67168220000010.5510.030.033
iFluor® 460 succinimidyl ester468493800001~0.810.980.46
iFluor® 440 succinimidyl ester4344804000010.6710.3520.229
iFluor® 665 succinimidyl ester667692110,00010.2210.120.09
iFluor® 690 succinimidyl ester68570422000010.3010.090.06
iFluor® 720 succinimidyl ester71674024000010.1410.150.13
iFluor® 740 succinimidyl ester74076422500010.2010.160.16
iFluor® 597 succinimidyl ester59861810000010.710.3350.514
iFluor® 770 succinimidyl ester77779725000010.160.090.08
iFluor® 780 succinimidyl ester78480825000010.1610.130.12
iFluor® 570 succinimidyl ester55757012000010.581--
iFluor® 830 succinimidyl ester830867----
iFluor® 675 succinimidyl ester683700---0.066
iFluor® 620 succinimidyl ester621636---0.04
iFluor® 605 succinimidyl ester603623----
iFluor® 625 succinimidyl ester624640----
iFluor® 510 succinimidyl ester511530----
iFluor® 540 succinimidyl ester540557---0.105
iFluor® 445 succinimidyl ester446558----
iFluor® 500 succinimidyl ester501520----
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Page updated on November 9, 2024

Ordering information

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Catalog Number
1040710407151871568
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Physical properties

Molecular weight

1257.45

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

Certificate of OriginDownload PDF
Intended useResearch Use Only (RUO)

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12171501
HeLa cells were incubated with (+ Tubulin) or without (-Tubulin) mouse anti-tubulin followed by iFluor®&nbsp;560 goat anti-mouse IgG conjugate stain and&nbsp;visualized with Cy3 Filter.
HeLa cells were incubated with (+ Tubulin) or without (-Tubulin) mouse anti-tubulin followed by iFluor®&nbsp;560 goat anti-mouse IgG conjugate stain and&nbsp;visualized with Cy3 Filter.
HeLa cells were incubated with (+ Tubulin) or without (-Tubulin) mouse anti-tubulin followed by iFluor®&nbsp;560 goat anti-mouse IgG conjugate stain and&nbsp;visualized with Cy3 Filter.
Spectral signature of iFluor® 560 dye. Data acquired on a 4-laser Cytek Aurora and normal human peripheral blood cells stained with clone SK3 (CD4) conjugated to iFluor® 560 dye (Cat. No. 100420A0) were used for analysis.

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