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iFluor® 440 succinimidyl ester

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Physical properties
Molecular weight692.83
SolventDMSO
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 yield0.671
Storage, safety and handling
Certificate of OriginDownload PDF
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12171501
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iFluor® 350 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 405 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 488 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 514 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 532 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 555 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 594 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 633 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 647 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 680 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 700 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 750 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 790 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 350 goat anti-rabbit IgG (H+L)
iFluor® 405 goat anti-rabbit IgG (H+L)
iFluor® 488 goat anti-rabbit IgG (H+L)
iFluor® 514 goat anti-rabbit IgG (H+L)
iFluor® 532 goat anti-rabbit IgG (H+L)
iFluor® 555 goat anti-rabbit IgG (H+L)
iFluor® 594 goat anti-rabbit IgG (H+L)
iFluor® 633 goat anti-rabbit IgG (H+L)
iFluor® 647 goat anti-rabbit IgG (H+L)
iFluor® 680 goat anti-rabbit IgG (H+L)
iFluor® 700 goat anti-rabbit IgG (H+L)
iFluor® 750 goat anti-rabbit IgG (H+L)
iFluor® 790 goat anti-rabbit IgG (H+L)
iFluor® 350 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 405 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 488 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 514 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 532 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 555 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 594 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 633 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 647 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 680 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 700 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 750 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
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iFluor® 350-streptavidin conjugate
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iFluor® 647-streptavidin conjugate
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iFluor® 750-streptavidin conjugate
iFluor® 750 RGD Conjugate
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iFluor® 405 hydrazide
iFluor® 800 acid
iFluor® 800 maleimide
iFluor® 810 acid
iFluor® 810 maleimide
iFluor® 820 acid
iFluor® 820 maleimide
iFluor® 860 acid
iFluor® 860 maleimide
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iFluor® 546 goat anti-mouse IgG (H+L) *Cross Adsorbed*
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iFluor® 568 maleimide
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iFluor® 568 Styramide *Superior Replacement for Alexa Fluor 568 tyramide*
iFluor® 594 Styramide *Superior Replacement for Alexa Fluor 594 tyramide*
iFluor® 647 Styramide *Superior Replacement for Alexa Fluor 647 tyramide*
iFluor® 680 Styramide *Superior Replacement for Alexa Fluor 680 tyramide and Opal 690*
iFluor® 700 Styramide *Superior Replacement for Alexa Fluor 700 tyramide*
iFluor® 750 Styramide *Superior Replacement for Alexa Fluor 750 tyramide*
iFluor® 790 Styramide *Superior Replacement for Alexa Fluor 790 tyramide*
iFluor® 555 Tyramide
iFluor® 647 Tyramide
iFluor® 350 PSA™ Imaging Kit with Goat Anti-Rabbit IgG
iFluor® 488 PSA™ Imaging Kit with Goat Anti-Rabbit IgG
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iFluor® 647 PSA™ Imaging Kit with Goat Anti-Mouse IgG
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iFluor® 820-streptavidin conjugate
iFluor® 840-streptavidin conjugate
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iFluor® 555 TCO
iFluor® 594 TCO
iFluor® 647 TCO
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iFluor® 555 Tetrazine
iFluor® 594 Tetrazine
iFluor® 647 Tetrazine
iFluor® 440-dUTP *1 mM in Tris Buffer (pH 7.5)*
iFluor® 665 maleimide
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iFluor® 514 Styramide *Superior Replacement for Opal 540*
iFluor® 532 Styramide
iFluor® 633 Styramide *Superior Replacement for Opal 650*
iFluor® 440 Styramide
iFluor® 460 Styramide
iFluor® 610 Styramide
iFluor® 660 Styramide
iFluor® 405 Styramide
iFluor®488-PEG12-dUTP *1 mM in Tris Buffer (pH 7.5)*
iFluor®555-PEG12-dUTP *1 mM in Tris Buffer (pH 7.5)*
iFluor®647-PEG12-dUTP *1 mM in Tris Buffer (pH 7.5)*
iFluor® 800 goat anti-mouse IgG (H+L)
iFluor® 800 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 810 goat anti-mouse IgG (H+L)
iFluor® 810 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 820 goat anti-mouse IgG (H+L)
iFluor® 820 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 840 goat anti-mouse IgG (H+L)
iFluor® 840 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 860 goat anti-mouse IgG (H+L)
iFluor® 860 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 800 goat anti-rabbit IgG (H+L)
iFluor® 800 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 810 goat anti-rabbit IgG (H+L)
iFluor® 810 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 820 goat anti-rabbit IgG (H+L)
iFluor® 820 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 840 goat anti-rabbit IgG (H+L)
iFluor® 840 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 860 goat anti-rabbit IgG (H+L)
iFluor® 860 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 430 Tyramide *Superior Replacement for Opal 480*
iFluor® 450 Tyramide *Superior Replacement for Opal 480*
iFluor® 546 maleimide
iFluor® 840 maleimide
iFluor® 770 maleimide
iFluor® 780 maleimide
iFluor® Ultra 594 succinimidyl ester
iFluor® Ultra 647 succinimidyl ester
iFluor® Ultra 750 succinimidyl ester
iFluor® 830 acid
iFluor® 830 maleimide
iFluor™ 405 azide
iFluor® 514 maleimide
iFluor® 660 maleimide
iFluor® 670 maleimide
iFluor® 560-dUTP *1 mM in Tris Buffer (pH 7.5)*
iFluor® 750-Concanavalin A Conjugate
iFluor® 605 maleimide
iFluor® 625 maleimide
iFluor® 510 maleimide
iFluor® 540 maleimide
iFluor® 350-Wheat Germ Agglutinin (WGA) Conjugate
iFluor® 532-Wheat Germ Agglutinin (WGA) Conjugate
iFluor® 680-Wheat Germ Agglutinin (WGA) Conjugate
iFluor® 700-Wheat Germ Agglutinin (WGA) Conjugate
iFluor® 750-Wheat Germ Agglutinin (WGA) Conjugate
iFluor® 790-Wheat Germ Agglutinin (WGA) Conjugate
iFluor® 570 Styramide *Superior Replacement for Alexa Fluor 568 tyramide*
iFluor® 670 Styramide *Replacement for Opal 690*
iFluor® 680 Tyramide *Superior Replacement for Opal 690*
iFluor™ 790 Azide
iFluor™ 790 Alkyne
iFluor® 720 maleimide
Show More (236)

OverviewpdfSDSpdfProtocol


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
  1. 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.
  2. 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.
  1. Prepare Sephadex G-25 column according to the manufacture instruction.
  2. Load the reaction mixture (From "Run conjugation reaction") to the top of the Sephadex G-25 column.
  3. Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top resin surface.
  4. 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 mg0.5 mg1 mg5 mg10 mg
1 mM144.336 µL721.678 µL1.443 mL7.217 mL14.434 mL
5 mM28.867 µL144.336 µL288.671 µL1.443 mL2.887 mL
10 mM14.434 µL72.168 µL144.336 µL721.678 µL1.443 mL

Molarity calculator

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

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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Spectrum


Open in Advanced Spectrum Viewer
spectrum

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 yield0.671

Product Family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
iFluor® 350 succinimidyl ester3454502000010.9510.830.23
iFluor® 405 succinimidyl ester4034273700010.9110.480.77
iFluor® 488 succinimidyl ester4915167500010.910.210.11
iFluor® 514 succinimidyl ester5115277500010.8310.2650.116
iFluor® 532 succinimidyl ester5375609000010.6810.260.16
iFluor® 555 succinimidyl ester55757010000010.6410.230.14
iFluor® 594 succinimidyl ester58760320000010.5310.050.04
iFluor® 633 succinimidyl ester64065425000010.2910.0620.044
iFluor® 647 succinimidyl ester65667025000010.2510.030.03
iFluor® 660 succinimidyl ester66367825000010.2610.070.08
iFluor® 680 succinimidyl ester68470122000010.2310.0970.094
iFluor® 700 succinimidyl ester69071322000010.2310.090.04
iFluor® 750 succinimidyl ester75777927500010.1210.0440.039
iFluor® 610 succinimidyl ester61062811000010.8510.320.49
iFluor® 710 succinimidyl ester71773919000010.6010.120.07
iFluor® 790 succinimidyl ester78781225000010.1310.10.09
iFluor® 800 succinimidyl ester80182025000010.1110.030.08
iFluor® 810 succinimidyl ester81182225000010.0510.090.15
iFluor® 820 succinimidyl ester82285025000010.110.16
iFluor® 860 succinimidyl ester85387825000010.10.14
iFluor® 546 succinimidyl ester54155710000010.6710.250.15
iFluor® 568 succinimidyl ester56858710000010.5710.340.15
iFluor® 430 succinimidyl ester4334984000010.7810.680.3
iFluor® 450 succinimidyl ester4515024000010.8210.450.27
iFluor® 840 succinimidyl ester8368792000001-0.20.09
iFluor® 560 succinimidyl ester56057112000010.5710.04820.069
iFluor® 670 succinimidyl ester67168220000010.5510.030.033
iFluor® 460 succinimidyl ester468493800001~0.810.980.46
iFluor® 665 succinimidyl ester667692110,00010.2210.120.09
iFluor® 690 succinimidyl ester68570422000010.3010.090.06
iFluor® 720 succinimidyl ester71674024000010.1410.150.13
iFluor® 740 succinimidyl ester74076422500010.2010.160.16
iFluor® 597 succinimidyl ester59861810000010.710.3350.514
iFluor® 770 succinimidyl ester77779725000010.160.090.08
iFluor® 780 succinimidyl ester78480825000010.1610.130.12
iFluor® 570 succinimidyl ester55757012000010.581--
iFluor® 830 succinimidyl ester830867----
iFluor® 675 succinimidyl ester683700---0.066
iFluor® 620 succinimidyl ester621636---0.04
iFluor® 605 succinimidyl ester603623----
iFluor® 625 succinimidyl ester624640----
iFluor® 510 succinimidyl ester511530----
iFluor® 540 succinimidyl ester540557---0.105
iFluor® 445 succinimidyl ester446558----
iFluor® 500 succinimidyl ester501520----
Show More (36)

References


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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