iFluor® 460 succinimidyl ester

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

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Physical properties

Molecular weight	793.99
Solvent	DMSO

Spectral properties

Correction Factor (260 nm)	0.98
Correction Factor (280 nm)	0.46
Extinction coefficient (cm ^-1 M ^-1)	80000¹
Excitation (nm)	468
Emission (nm)	493
Quantum yield	~0.8¹

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

Alternative formats

iFluor® 460 maleimide

Overview SDS Protocol

Molecular weight

793.99

Correction Factor (260 nm)

0.98

Correction Factor (280 nm)

0.46

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

80000¹

Excitation (nm)

468

Emission (nm)

493

Quantum yield

~0.8¹

AAT Bioquest's iFluor® dyes are optimized for labeling proteins, particularly antibodies. These dyes are bright, photostable, and have minimal quenching on proteins. Although the 460 nm blue diode laser is being installed in numerous new fluorescence instruments, few dyes can be well excited at 460 nm. iFluor® 460 is optimized to be well excited by the blue diode laser at 460 nm, enabling new biological applications for the new fluorescence instruments equipped with the 460 nm blue diode laser. iFluor® 460 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™ 460 SE stock solution (Solution B)

Add anhydrous DMSO into the vial of iFluor™ 460 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™ 460 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® 460 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	125.946 µL	629.731 µL	1.259 mL	6.297 mL	12.595 mL
5 mM	25.189 µL	125.946 µL	251.892 µL	1.259 mL	2.519 mL
10 mM	12.595 µL	62.973 µL	125.946 µL	629.731 µL	1.259 mL

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

Correction Factor (260 nm)	0.98
Correction Factor (280 nm)	0.46
Extinction coefficient (cm ^-1 M ^-1)	80000¹
Excitation (nm)	468
Emission (nm)	493
Quantum yield	~0.8¹

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® 647 succinimidyl ester	656	670	250000¹	0.25¹	0.03	0.03
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® 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	557	570	120000¹	0.58¹	-	-
iFluor® 830 succinimidyl ester	830	867	-	-	-	-
iFluor® 605 succinimidyl ester	562	623	-	-	-	-
iFluor® 625 succinimidyl ester	575	640	-	-	-	-
iFluor® 510 succinimidyl ester	511	530	-	-	-	-
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References

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Real-time in vivo imaging of extracellular ATP in the brain with a hybrid-type fluorescent sensor.
Authors: Kitajima, Nami and Takikawa, Kenji and Sekiya, Hiroshi and Satoh, Kaname and Asanuma, Daisuke and Sakamoto, Hirokazu and Takahashi, Shodai and Hanaoka, Kenjiro and Urano, Yasuteru and Namiki, Shigeyuki and Iino, Masamitsu and Hirose, Kenzo
Journal: eLife (2020)

Challenging a Preconception: Optoacoustic Spectrum Differs from the Optical Absorption Spectrum of Proteins and Dyes for Molecular Imaging.
Authors: Fuenzalida Werner, Juan Pablo and Huang, Yuanhui and Mishra, Kanuj and Janowski, Robert and Vetschera, Paul and Heichler, Christina and Chmyrov, Andriy and Neufert, Clemens and Niessing, Dierk and Ntziachristos, Vasilis and Stiel, Andre C
Journal: Analytical chemistry (2020)

Nanobody click chemistry for convenient site-specific fluorescent labelling, single step immunocytochemistry and delivery into living cells by photoporation and live cell imaging.
Authors: Hebbrecht, Tim and Liu, Jing and Zwaenepoel, Olivier and Boddin, Gaëlle and Van Leene, Chloé and Decoene, Klaas and Madder, Annemieke and Braeckmans, Kevin and Gettemans, Jan
Journal: New biotechnology (2020): 33-43

Noninvasive In Situ Ratiometric Imaging of Biometals Based on Self-Assembled Peptide Nanoribbon.
Authors: Lei, Li and Li, Min and Wu, Sufen and Xu, Zhiai and Geng, Ping and Tian, Yang and Fu, Ying and Zhang, Wen
Journal: Analytical chemistry (2020): 5838-5845

Near-Infrared In Vivo Whole-Body Fluorescence Imaging of PNA.
Authors: Lim, Ernest Wee Kiat and Brolin, Camilla and Nielsen, Peter E
Journal: Methods in molecular biology (Clifton, N.J.) (2020): 251-260

Identification and local manipulation of bone marrow vasculature during intravital imaging.
Authors: Morikawa, Takayuki and Tamaki, Shinpei and Fujita, Shinya and Suematsu, Makoto and Takubo, Keiyo
Journal: Scientific reports (2020): 6422

A novel tracer for in vivo optical imaging of fatty acid metabolism in the heart and brown adipose tissue.
Authors: Panagia, Marcello and Yang, Jing and Gale, Eric and Wang, Huan and Luptak, Ivan and Chen, Howard H and Patel, Dakshesh and Croteau, Dominique and Pimentel, David Richard and Bachschmid, Markus Michael and Colucci, Wilson S and Ran, Chongzhao and Sosnovik, David E
Journal: Scientific reports (2020): 11209

A novel two-photon ratiometric fluorescent probe for imaging and sensing of BACE1 in different regions of AD mouse brain.
Authors: Ge, Lihong and Liu, Zhichao and Tian, Yang
Journal: Chemical science (2020): 2215-2224

Preliminary study on the application of en bloc resection combined with near-infrared molecular imaging technique in the diagnosis and treatment of bladder cancer.
Authors: Yang, Yongjun and Yang, Xiaofeng and Liu, Chao and Li, Jiawei
Journal: World journal of urology (2020)

CD24-targeted fluorescence imaging in patient-derived xenograft models of high-grade serous ovarian carcinoma.
Authors: Kleinmanns, Katrin and Bischof, Katharina and Anandan, Shamundeeswari and Popa, Mihaela and Akslen, Lars A and Fosse, Vibeke and Karlsen, Ida Tveit and Gjertsen, Bjørn T and Bjørge, Line and McCormack, Emmet
Journal: EBioMedicine (2020): 102782