iFluor® 647 succinimidyl ester

HeLa cells were stained with mouse anti-tubulin followed with iFluor<sup>TM</sup> 647 goat anti-mouse IgG (H+L) (red); and nuclei were stained with DAPI (blue).

HeLa cells were incubated with mouse anti-tubulin followed by AAT’s iFluor<sup>TM</sup> 647 goat anti-mouse IgG conjugate (Red, Left) or Alexa Fluor<sup>®</sup> 647 goat anti-mouse IgG<sup>  </sup>(Red, Right), respectively. Cell nuclei were stained with Hoechst 33342 (Blue, Cat#17530).

HeLa cells were incubated with mouse anti-tubulin and biotin goat anti-mouse IgG followed by AAT’s iFluor® 647-streptavidin conjugate (Red, Left) or streptavidin conjugated with Alexa Fluor<sup>®</sup> 647 (Red, Right), respectively.

Fluorescence In Situ Hybridization of Cy5 and iFluor® 647-dUTP labelled Telomere probes in metaphase HeLa cells.

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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	1274.66
Solvent	DMSO

Spectral properties

Correction Factor (260 nm)	0.03
Correction Factor (280 nm)	0.03
Correction Factor (656 nm)	0.0793
Extinction coefficient (cm ^-1 M ^-1)	250000¹
Excitation (nm)	656
Emission (nm)	670
Quantum yield	0.25¹

Storage, safety and handling

Certificate of Origin	Download PDF
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

Alternative formats

iFluor® 647 amine

iFluor® 647 maleimide

iFluor® 647 azide

iFluor® 647 alkyne

iFluor® 647 hydrazide

Overview SDS Protocol

Molecular weight

1274.66

Correction Factor (260 nm)

0.03

Correction Factor (280 nm)

0.03

Correction Factor (656 nm)

0.0793

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

250000¹

Excitation (nm)

656

Emission (nm)

670

Quantum yield

0.25¹

AAT Bioquest's iFluor® dyes are optimized for labeling proteins, particularly antibodies. These dyes are bright, photostable, and have minimal quenching on proteins. They can be well excited by the major laser lines of fluorescence instruments (e.g., 350, 405, 488, 555, and 633 nm). The iFluor® 647 family has spectral properties essentially identical to those of Cy5®. Compared to Cy5® probes, iFluor® 647 reagents have much stronger fluorescence and higher photostability. Their fluorescence is pH-independent from pH 3 to 11. These spectral characteristics make this new dye family an excellent alternative to Cy5® and Alexa Fluor® 647 (Cy5® and Alexa Fluor® are the trademarks of GE Health Care and Invitrogen). iFluor® 647 SE is reasonably stable and shows good reactivity and selectivity with protein amino groups.

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™ 647 SE stock solution (Solution B)

Add anhydrous DMSO into the vial of iFluor™ 647 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™ 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 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® 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	78.452 µL	392.261 µL	784.523 µL	3.923 mL	7.845 mL
5 mM	15.69 µL	78.452 µL	156.905 µL	784.523 µL	1.569 mL
10 mM	7.845 µL	39.226 µL	78.452 µL	392.261 µL	784.523 µL

Molarity calculator

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

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
	/		=		x		=

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Correction Factor (260 nm)	0.03
Correction Factor (280 nm)	0.03
Correction Factor (656 nm)	0.0793
Extinction coefficient (cm ^-1 M ^-1)	250000¹
Excitation (nm)	656
Emission (nm)	670
Quantum yield	0.25¹

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield	Correction Factor (260 nm)	Correction Factor (280 nm)
iFluor® 350 succinimidyl ester	345	450	20000¹	0.95¹	0.83	0.23
iFluor® 405 succinimidyl ester	403	427	37000¹	0.91¹	0.48	0.77
iFluor® 488 succinimidyl ester	491	516	75000¹	0.9¹	0.21	0.11
iFluor® 514 succinimidyl ester	511	527	75000¹	0.83¹	0.265	0.116
iFluor® 532 succinimidyl ester	537	560	90000¹	0.68¹	0.26	0.16
iFluor® 555 succinimidyl ester	557	570	100000¹	0.64¹	0.23	0.14
iFluor® 594 succinimidyl ester	588	604	180000¹	0.53¹	0.05	0.04
iFluor® 633 succinimidyl ester	640	654	250000¹	0.29¹	0.062	0.044
iFluor® 660 succinimidyl ester	663	678	250000¹	0.26¹	0.07	0.08
iFluor® 680 succinimidyl ester	684	701	220000¹	0.23¹	0.097	0.094
iFluor® 700 succinimidyl ester	690	713	220000¹	0.23¹	0.09	0.04
iFluor® 750 succinimidyl ester	757	779	275000¹	0.12¹	0.044	0.039
iFluor® 610 succinimidyl ester	610	628	110000¹	0.85¹	0.32	0.49
iFluor® 710 succinimidyl ester	717	739	190000¹	0.60¹	0.12	0.07
iFluor® 790 succinimidyl ester	787	812	250000¹	0.13¹	0.1	0.09
iFluor® 800 succinimidyl ester	801	820	250000¹	0.11¹	0.03	0.08
iFluor® 810 succinimidyl ester	811	822	250000¹	0.05¹	0.09	0.15
iFluor® 820 succinimidyl ester	822	850	250000¹		0.11	0.16
iFluor® 860 succinimidyl ester	853	878	250000¹		0.1	0.14
iFluor® 546 succinimidyl ester	541	557	100000¹	0.67¹	0.25	0.15
iFluor® 568 succinimidyl ester	568	587	100000¹	0.57¹	0.34	0.15
iFluor® 430 succinimidyl ester	433	498	40000¹	0.78¹	0.68	0.3
iFluor® 450 succinimidyl ester	451	502	40000¹	0.82¹	0.45	0.27
iFluor® 840 succinimidyl ester	836	879	200000¹	-	0.2	0.09
iFluor® 560 succinimidyl ester	560	571	120000¹	0.57¹	0.0482	0.069
iFluor® 670 succinimidyl ester	671	682	200000¹	0.55¹	0.03	0.033
iFluor® 460 succinimidyl ester	468	493	80000¹	~0.8¹	0.98	0.46
iFluor® 440 succinimidyl ester	434	480	40000¹	0.67¹	0.352	0.229
iFluor® 665 succinimidyl ester	667	692	110,000¹	0.22¹	0.12	0.09
iFluor® 690 succinimidyl ester	685	704	220000¹	0.30¹	0.09	0.06
iFluor® 720 succinimidyl ester	716	740	240000¹	0.14¹	0.15	0.13
iFluor® 740 succinimidyl ester	742	764	225000¹	0.20¹	0.16	0.16
iFluor® 597 succinimidyl ester	598	618	100000¹	0.7¹	0.335	0.514
iFluor® 770 succinimidyl ester	777	797	250000¹	0.16	0.09	0.08
iFluor® 780 succinimidyl ester	784	808	250000¹	0.16¹	0.13	0.12
iFluor® 570 succinimidyl ester	560	571	120000¹	0.58¹	0.048	0.069
iFluor® 830 succinimidyl ester	830	867	-	-	-	-
zFluor™ 647 succinimidyl ester	648	668	-	-	-	-
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Images

Figure 1. HeLa cells were stained with mouse anti-tubulin followed with iFluor^TM 647 goat anti-mouse IgG (H+L) (red); and nuclei were stained with DAPI (blue).

Figure 2. HeLa cells were incubated with mouse anti-tubulin followed by AAT’s iFluor^TM 647 goat anti-mouse IgG conjugate (Red, Left) or Alexa Fluor^® 647 goat anti-mouse IgG(Red, Right), respectively. Cell nuclei were stained with Hoechst 33342 (Blue, Cat#17530).

Figure 3. HeLa cells were incubated with mouse anti-tubulin and biotin goat anti-mouse IgG followed by AAT’s iFluor® 647-streptavidin conjugate (Red, Left) or streptavidin conjugated with Alexa Fluor^® 647 (Red, Right), respectively.

Figure 4. Fluorescence In Situ Hybridization of Cy5 and iFluor® 647-dUTP labelled Telomere probes in metaphase HeLa cells.

Citations

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The cell adhesion receptor TMIGD1 recruits Scribble to the basolateral membrane via direct interaction
Authors: Ebnet, Klaus and Th{\"u}ring, Eva-Maria and Hartmann, Christian and Maddumage, Janesha and Javorsky, Airah and Michels, Birgitta and Gerke, Volker and Banks, Lawrence and Humbert, Patrick and Kvansakul, Marc
Journal: (2023)

Inhibition of MYC suppresses programmed cell death ligand-1 expression and enhances immunotherapy in triple-negative breast cancer
Authors: Li, Xintong and Tang, Lin and Chen, Qin and Cheng, Xumin and Liu, Yiqiu and Wang, Cenzhu and Zhu, Chengjun and Xu, Kun and Gao, Fangyan and Huang, Jinyi and others,
Journal: Chinese Medical Journal (2022): 10--1097

Immune-regulating bimetallic metal-organic framework nanoparticles designed for cancer immunotherapy
Authors: Dai, Zan and Wang, Qiaoyun and Tang, Jie and Wu, Min and Li, Haoze and Yang, Yannan and Zhen, Xu and Yu, Chengzhong
Journal: Biomaterials (2022): 121261

Preparation and identification of monoclonal antibodies against porcine CD103
Authors: Zhang, Tao and Yu, Haoyuan and Aryal, Manita and Yang, Jing and Li, Maolin and Li, Shuxian and Zhang, Na and Shi, Han and Li, Baoyu and Liu, Guangliang and others,
Journal: Applied Microbiology and Biotechnology (2022): 1--11

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

Specificity of TGF-$\beta$1 signal designated by LRRC33 and integrin $\alpha$V$\beta$8
Authors: Duan, Zelin and Lin, Xuezhen and Wang, Lixia and Zhen, Qiuxin and Jiang, Yuefeng and Chen, Chuxin and Yang, Jing and Lee, Chia-Hsueh and Qin, Yan and Li, Ying and others,
Journal: Nature Communications (2022): 4988

A nano-innate immune system activator for cancer therapy in a 4T1 tumor-bearing mouse model
Authors: Liu, Xiang-Yu and Zhu, Mao-Hua and Wang, Xiao-Yu and Dong, Xiao and Liu, Hai-Jun and Li, Rui-Yang and Jia, Shi-Chong and Lu, Qin and Zhao, Mei and Sun, Peng and others,
Journal: Journal of nanobiotechnology (2022): 1--15

Poly ADP-ribosylation of SET8 leads to aberrant H4K20 methylation in mammalian nuclear genome
Authors: Est{\`e}ve, Pierre-Olivier and Sen, Sagnik and Vishnu, Udayakumar S and Ruse, Cristian and Chin, Hang Gyeong and Pradhan, Sriharsa
Journal: Communications biology (2022): 1--18

The PARP1 Inhibitor Niraparib Represses DNA Damage Repair and Synergizes with Temozolomide for Antimyeloma Effects
Authors: Shen, Hong-Yuan and Tang, Hai-Long and Zheng, Yan-Hua and Feng, Juan and Dong, Bao-Xia and Chen, Xie-Qun
Journal: Journal of Oncology (2022)

A Substance P (SP)/Neurokinin-1 Receptor 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: Journal of Immunology Research (2022)

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