iFluor® 440 succinimidyl ester

Ordering information

Price
Catalog Number
Unit Size
Quantity

Add to cart

Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
International	See distributors
Bulk request	Inquire
Custom size	Inquire
Shipping	Standard overnight for United States, inquire for international

Physical properties

Molecular weight	692.83
Solvent	DMSO

Spectral properties

Absorbance (nm)	430
Correction Factor (260 nm)	0.352
Correction Factor (280 nm)	0.229
Extinction coefficient (cm ^-1 M ^-1)	40000¹
Excitation (nm)	434
Emission (nm)	480
Quantum yield	0.67¹

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

Overview SDS Protocol

Molecular weight

692.83

Absorbance (nm)

430

Correction Factor (260 nm)

0.352

Correction Factor (280 nm)

0.229

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

40000¹

Excitation (nm)

434

Emission (nm)

480

Quantum yield

0.67¹

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). iFluor® 440 dyes are designed to be a superior replacement for DEAC dye that has extremely poor water solubility. iFluor® 440 SE is reasonably stable and shows good reactivity and selectivity with protein amino groups. Under the same conditions, iFluor® 440 dye conjugates are significantly brighter than the corresponding bioconjugates of DEAC or other spectrally similar dyes (such as SpectrumAqua), making the iFluor® 440 conjugates much more sensitive.

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™ 440 SE stock solution (Solution B)

Add anhydrous DMSO into the vial of iFluor™ 440 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™ 440 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® 440 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	144.336 µL	721.678 µL	1.443 mL	7.217 mL	14.434 mL
5 mM	28.867 µL	144.336 µL	288.671 µL	1.443 mL	2.887 mL
10 mM	14.434 µL	72.168 µL	144.336 µL	721.678 µL	1.443 mL

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

Absorbance (nm)	430
Correction Factor (260 nm)	0.352
Correction Factor (280 nm)	0.229
Extinction coefficient (cm ^-1 M ^-1)	40000¹
Excitation (nm)	434
Emission (nm)	480
Quantum yield	0.67¹

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® 460 succinimidyl ester	468	493	80000¹	~0.8¹	0.98	0.46
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	740	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® 675 succinimidyl ester	683	700	-	-	-	0.066
iFluor® 620 succinimidyl ester	621	636	-	-	-	0.04
iFluor® 605 succinimidyl ester	603	623	-	-	-	-
iFluor® 625 succinimidyl ester	624	640	-	-	-	-
iFluor® 510 succinimidyl ester	511	530	-	-	-	-
iFluor® 540 succinimidyl ester	540	557	-	-	-	0.105
iFluor® 445 succinimidyl ester	446	558	-	-	-	-
Show More (35)

References

View all 12 references: Citation Explorer

7-(Diethylamino)coumarin-3-carboxylic acid as derivatization reagent for 405 nm laser-induced fluorescence detection: A case study for the analysis of sulfonamides by capillary electrophoresis.
Authors: Wu, Chengxin and Sun, Yuanyuan and Wang, Yuanhang and Duan, Wenzhen and Hu, Jiangyue and Zhou, Lei and Pu, Qiaosheng
Journal: Talanta (2019): 16-22

Design, synthesis and perception of fluorescently labeled isoprenoid cytokinins.
Authors: Kubiasová, Karolina and Mik, Václav and Nisler, Jaroslav and Hönig, Martin and Husičková, Alexandra and Spíchal, Lukáš and Pěkná, Zuzana and Šamajová, Olga and Doležal, Karel and Plíhal, Ondřej and Benková, Eva and Strnad, Miroslav and Plíhalová, Lucie
Journal: Phytochemistry (2018): 1-11

Intracellular Uncaging of cGMP with Blue Light.
Authors: Agarwal, Hitesh K and Zhai, Shenyu and Surmeier, D James and Ellis-Davies, Graham C R
Journal: ACS chemical neuroscience (2017): 2139-2144

Wavelength-selective one- and two-photon uncaging of GABA.
Authors: Amatrudo, Joseph M and Olson, Jeremy P and Lur, G and Chiu, Chiayu Q and Higley, Michael J and Ellis-Davies, Graham C R
Journal: ACS chemical neuroscience (2014): 64-70

A water soluble fluorescent polymer as a dual colour sensor for temperature and a specific protein.
Authors: Inal, Sahika and Kölsch, Jonas D and Sellrie, Frank and Schenk, Jörg A and Wischerhoff, Erik and Laschewsky, André and Neher, Dieter
Journal: Journal of materials chemistry. B (2013): 6373-6381

Structures, synthesis, and human Nod1 stimulation of immunostimulatory bacterial peptidoglycan fragments in the environment.
Authors: Fujimoto, Yukari and Fukase, Koichi
Journal: Journal of natural products (2011): 518-25

Fluorescent agonists for the Torpedo nicotinic acetylcholine receptor.
Authors: Krieger, Florian and Mourot, Alexandre and Araoz, Romulo and Kotzyba-Hibert, Florence and Molgó, Jordi and Bamberg, Ernst and Goeldner, Maurice
Journal: Chembiochem : a European journal of chemical biology (2008): 1146-53

Antimicrobial activity of various cationic molecules on foodborne pathogens.
Authors: Conte, Mariachiara and Aliberti, Francesco and Fucci, Laura and Piscopo, Marina
Journal: World journal of microbiology & biotechnology (2007): 1679-83

A series of related nucleotide analogues that aids optimization of fluorescence signals in probing the mechanism of P-loop ATPases, such as actomyosin.
Authors: Webb, Martin R and Reid, Gordon P and Munasinghe, V Ranjit N and Corrie, John E T
Journal: Biochemistry (2004): 14463-71

Fluorescent coumarin-labeled nucleotides to measure ADP release from actomyosin.
Authors: Webb, M R and Corrie, J E
Journal: Biophysical journal (2001): 1562-9