iFluor® Ultra 647 succinimidyl ester

HeLa cells were incubated with mouse anti-tubulin followed by AAT’s iFluor<sup>TM</sup> Ultra 647 goat anti-mouse IgG conjugate or Alexa Fluor<sup>®</sup> 647 goat anti-mouse IgG.

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Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
Quotation	Request
International	See distributors
Shipping	Standard overnight for United States, inquire for international

Physical properties

Molecular weight	2634.28
Solvent	DMSO

Spectral properties

Absorbance (nm)	654
Correction Factor (260 nm)	0.07
Correction Factor (280 nm)	0.07
Extinction coefficient (cm ^-1 M ^-1)	250000¹
Excitation (nm)	655
Emission (nm)	670
Quantum yield	0.39¹

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

2634.28

Absorbance (nm)

654

Correction Factor (260 nm)

0.07

Correction Factor (280 nm)

0.07

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

250000¹

Excitation (nm)

655

Emission (nm)

670

Quantum yield

0.39¹

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 647 are far superior to the conjugates of other existing similar dyes such as Cy5, Dylight 650 and Alexa Fluor® 647. iFluor® Ultra 647 conjugates are significantly brighter than the conjugates prepared with Cy5, Dylight 650 and Alexa Fluor® 647 under the same conditions. Additionally, the fluorescence of iFluor® Ultra 647 is not affected by pH (4-10). iFluor® Ultra 647 SE dye is reasonably stable and shows good reactivity and selectivity with protein amino groups. iFluor® Ultra 647 has spectral properties and reactivity similar to Cy5, Dylight 650 and Alexa Fluor® 647 (Cy5® and Alexa Fluor® is the trademarks of GE Healthcare and ThermoFisher respectively).

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 ~8.5) 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 647 SE stock solution (Solution B)

Add 100 µL high-quality, anhydrous dimethylsulfoxide (DMSO) or dimethyl-formamide (DMF) to 1 mg iFluor™ Ultra 647 SE to prepare 10 mg/mL 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.
Note Once reconstituted, the NHS ester reactive dye solution is not very stable, especially if exposed to moisture. It could hydrolyze into the nonreactive free acid in aqueous solutions.

SAMPLE EXPERIMENTAL PROTOCOL

This labeling protocol was developed for the conjugate of Goat anti-mouse IgG with iFluor™ Ultra 647 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 6-10 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.
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 with 15 mL MWCO=30K filter (https://www.emdmillipore.com/US/en/product/Amicon-Ultra-4-Centrifugal-Filter-Units,MM_NF-C7719)

Prepare column according to the manufacture instruction.
Load the reaction mixture (From "Run conjugation reaction") to the top of the column.
Add 4 mL of PBS (pH 7.2-7.4) as soon as the sample runs.
Centrifuge to concentrate to ~0.4 mL.
Add 4 mL PBS and then concentrate to ~0.4 mL.
Repeat ~3 times, until the elution absorbance at 650 nm < 0.1.
Collect the purified Antibody-iFluor™Ultra 647 conjugate solution.

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of iFluor® Ultra 647 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	37.961 µL	189.805 µL	379.61 µL	1.898 mL	3.796 mL
5 mM	7.592 µL	37.961 µL	75.922 µL	379.61 µL	759.221 µL
10 mM	3.796 µL	18.981 µL	37.961 µL	189.805 µL	379.61 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

Open in Advanced Spectrum Viewer

Spectral properties

Absorbance (nm)	654
Correction Factor (260 nm)	0.07
Correction Factor (280 nm)	0.07
Extinction coefficient (cm ^-1 M ^-1)	250000¹
Excitation (nm)	655
Emission (nm)	670
Quantum yield	0.39¹

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 594 succinimidyl ester	586	601	180000¹	0.91¹	0.07	0.05
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 647 goat anti-mouse IgG conjugate or Alexa Fluor^® 647 goat anti-mouse IgG.

Citations

View all 5 citations: Citation Explorer

Site-specific labeling and functional efficiencies of human fibroblast growth Factor-1 with a range of fluorescent Dyes in the flexible N-Terminal region and a rigid $\beta$-turn region
Authors: Mohale, Mamello and Gundampati, Ravi Kumar and Kumar, Thallapuranam Krishnaswamy Suresh and Heyes, Colin D
Journal: Analytical biochemistry (2022): 114524

SP/NK-1R Axis Promotes Perineural Invasion of Pancreatic Cancer and is Affected by lncRNA LOC389641
Authors: Ji, Tengfei and Ma, Keqiang and Wu, Hongsheng and Cao, Tiansheng
Journal: (2021)

Efferocytosis induces macrophage proliferation to help resolve tissue injury
Authors: Gerlach, Brennan D and Ampomah, Patrick B and Yurdagul Jr, Arif and Liu, Chuang and Lauring, Max C and Wang, Xiaobo and Kasikara, Canan and Kong, Na and Shi, Jinjun and Tao, Wei and others,
Journal: Cell metabolism (2021): 2445--2463

Pharmacological targeting of Sam68 functions in colorectal cancer stem cells
Authors: Masibag, Angelique N and Bergin, Christopher J and Haebe, Joshua R and Zouggar, A{\"\i}cha and Shah, Muhammad S and Sandouka, Tamara and da Silva, Amanda Mendes and Desrochers, Fran{\c{c}}ois M and Fournier-Morin, Aube and Benoit, Yannick D
Journal: Iscience (2021): 103442

Influence of particle geometry on gastrointestinal transit and absorption following oral administration
Authors: Li, Dong and Zhuang, Jie and He, Haisheng and Jiang, Sifan and Banerjee, Amrita and Lu, Yi and Wu, Wei and Mitragotri, Samir and Gan, Li and Qi, Jianping
Journal: ACS applied materials \& interfaces (2017): 42492--42502

References

View all 50 references: Citation Explorer

Comparison of separation modes for microchip electrophoresis of proteins.
Authors: Samarasinghe, Thushara N and Zeng, Yong and Johnson, Carey K
Journal: Journal of separation science (2021): 744-751

Small molecule microarray identifies inhibitors of tyrosyl-DNA phosphodiesterase 1 that simultaneously access the catalytic pocket and two substrate binding sites.
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Journal: Chemical science (2021): 3876-3884

Investigating Single-Molecule Fluorescence Spectral Heterogeneity of Rhodamines Using High-Throughput Single-Molecule Spectroscopy.
Authors: Zhang, Yang and Zhang, Yu and Song, Ki-Hee and Lin, Wei and Sun, Cheng and Schatz, George C and Zhang, Hao F
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Targeted volumetric single-molecule localization microscopy of defined presynaptic structures in brain sections.
Authors: Pauli, Martin and Paul, Mila M and Proppert, Sven and Mrestani, Achmed and Sharifi, Marzieh and Repp, Felix and Kürzinger, Lydia and Kollmannsberger, Philip and Sauer, Markus and Heckmann, Manfred and Sirén, Anna-Leena
Journal: Communications biology (2021): 407

Intravital Imaging of Circulating Red Blood Cells in the Retinal Vasculature of Growing Mice.
Authors: Jeon, Jehwi and Hwang, Yoonha and Lee, Jingu and Kong, Eunji and Moon, Jieun and Hong, Sujung and Kim, Pilhan
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A Combined Immunofluorescence and Fluorescent Viability Cocktail Staining Procedure for Rapid Microscopic Detection and Enumeration of Live Legionella pneumophila.
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Multimodality labeling of NGR-functionalized hyaluronan for tumor targeting and radiotherapy.
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