iFluor® Ultra 594 succinimidyl ester

HeLa cells were incubated with mouse anti-tubulin followed by AAT’s iFluor<sup>TM</sup> Ultra 594 goat anti-mouse IgG conjugate or Alexa Fluor<sup>®</sup> 594 goat anti-mouse IgG.

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Physical properties

Molecular weight	1436.72
Solvent	DMSO

Spectral properties

Absorbance (nm)	585
Correction Factor (260 nm)	0.07
Correction Factor (280 nm)	0.05
Extinction coefficient (cm ^-1 M ^-1)	180000¹
Excitation (nm)	586
Emission (nm)	601
Quantum yield	0.91¹

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12171501

Overview SDS Protocol

Molecular weight

1436.72

Absorbance (nm)

585

Correction Factor (260 nm)

0.07

Correction Factor (280 nm)

0.05

Extinction coefficient (cm ^-1 M ^-1)

180000¹

Excitation (nm)

586

Emission (nm)

601

Quantum yield

0.91¹

Fluorescent dye-conjugated antibodies provide a tool for identifying proteins in many applications including fluorescent cell imaging, flow cytometry, western blotting, immunohistochemistry and more. The advantages of using a fluorescently labeled antibody include higher sensitivity, multiplexing capabilities, and ease of use. iFluor® Ultra family is a recent upgrade of our popular iFluor® dyes and optimized for labeling antibodies used for fluorescence imaging and flow cytometry applications. Antibody conjugates prepared with iFluor® Ultra 594 are far superior to the conjugates of other existing similar dyes such as Alexa Fluor® 594. iFluor® Ultra 594 conjugates are significantly brighter than the conjugates prepared with Alexa Fluor® 594 under the same conditions. Additionally, the fluorescence of iFluor® Ultra 594 is not affected by pH (4-10). iFluor® Ultra 594 SE dye is reasonably stable and shows good reactivity and selectivity with protein amino groups. iFluor® Ultra 594 has spectral properties and reactivity similar to Alexa Fluor® 594 (Alexa Fluor® is the trademark of ThermoFisher).

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™ Ultra 594 SE stock solution (Solution B)

Add anhydrous DMSO into the vial of iFluor™ Ultra 594 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™ Ultra 594 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

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.
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.

Prepare Sephadex G-25 column according to the manufacture instruction.
Load the reaction mixture (From "Run conjugation reaction") to the top of the Sephadex G-25 column.
Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top resin surface.
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® Ultra 594 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 mg	0.5 mg	1 mg	5 mg	10 mg
1 mM	69.603 µL	348.015 µL	696.03 µL	3.48 mL	6.96 mL
5 mM	13.921 µL	69.603 µL	139.206 µL	696.03 µL	1.392 mL
10 mM	6.96 µL	34.801 µL	69.603 µL	348.015 µL	696.03 µL

Molarity calculator

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

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
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Spectrum

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Spectral properties

Absorbance (nm)	585
Correction Factor (260 nm)	0.07
Correction Factor (280 nm)	0.05
Extinction coefficient (cm ^-1 M ^-1)	180000¹
Excitation (nm)	586
Emission (nm)	601
Quantum yield	0.91¹

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield	Correction Factor (260 nm)	Correction Factor (280 nm)
iFluor® Ultra 647 succinimidyl ester	655	670	250000¹	0.39¹	0.07	0.07
iFluor® Ultra 750 succinimidyl ester	749	773	250000¹	0.32¹	0.04	0.05

Images

Figure 1. HeLa cells were incubated with mouse anti-tubulin followed by AAT’s iFluor^TM Ultra 594 goat anti-mouse IgG conjugate or Alexa Fluor^® 594 goat anti-mouse IgG.

References

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Journal: European journal of pharmacology (2021): 174035

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Journal: Angewandte Chemie (International ed. in English) (2021)

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Journal: Cephalalgia : an international journal of headache (2020): 229-240

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Authors: Moarefian, Maryam and Davalos, Rafael V and Tafti, Danesh K and Achenie, Luke E and Jones, Caroline N
Journal: Lab on a chip (2020): 3310-3321

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Authors: Zhang, Kun and Li, Bin and Li, Peifang and Yang, Xiaoli and Cui, Huixian and Liu, Xiaoyun
Journal: Acta neurobiologiae experimentalis (2019): 302-308

Super blinking and biocompatible nanoprobes based on dye doped BSA nanoparticles for super resolution imaging.
Authors: Zong, Shenfei and Pan, Fengmei and Zhang, Ruohu and Chen, Chen and Wang, Zhuyuan and Cui, Yiping
Journal: Nanotechnology (2019): 065701

[Sepsis impairs aggregation of nicotinic acetylcholine receptors on murine skeletal muscle cell membranes by inhibiting AKT/GSK3β phosphorylation].
Authors: Li, Tianmei and Liu, Li and Wang, Xiaobin
Journal: Nan fang yi ke da xue xue bao = Journal of Southern Medical University (2019): 1337-1343