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iFluor® Ultra 594 succinimidyl ester

HeLa cells were incubated with mouse anti-tubulin followed by AAT’s iFluor<sup>TM</sup> Ultra 594 goat anti-mouse IgG conjugate or Alexa Fluor<sup>®</sup> 594 goat anti-mouse IgG.
HeLa cells were incubated with mouse anti-tubulin followed by AAT’s iFluor<sup>TM</sup> Ultra 594 goat anti-mouse IgG conjugate or Alexa Fluor<sup>®</sup> 594 goat anti-mouse IgG.
Ordering information
Price ()
Catalog Number71650
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Physical properties
Molecular weight1436.72
SolventDMSO
Spectral properties
Absorbance (nm)585
Correction Factor (260 nm)0.07
Correction Factor (280 nm)0.05
Extinction coefficient (cm -1 M -1)1800001
Excitation (nm)586
Emission (nm)601
Quantum yield0.911
Storage, safety and handling
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® 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*
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iFluor® 350-streptavidin conjugate
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iFluor® 750-streptavidin conjugate
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iFluor® 405 hydrazide
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iFluor® 568 maleimide
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iFluor® 555 Styramide *Superior Replacement for Alexa Fluor 555 tyramide and Opal 570*
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*
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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*
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iFluor® 450 Tyramide *Superior Replacement for Opal 480*
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iFluor® 770 maleimide
iFluor® 780 maleimide
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iFluor® 647 succinimidyl ester
iFluor® 660 succinimidyl ester
iFluor® 680 succinimidyl ester
iFluor® 700 succinimidyl ester
iFluor® 750 succinimidyl ester
iFluor® 610 succinimidyl ester
iFluor® 710 succinimidyl ester
iFluor® 790 succinimidyl ester
iFluor® 800 succinimidyl ester
iFluor® 810 succinimidyl ester
iFluor® 820 succinimidyl ester
iFluor® 860 succinimidyl ester
iFluor® 546 succinimidyl ester
iFluor® 568 succinimidyl ester
iFluor® 430 succinimidyl ester
iFluor® 450 succinimidyl ester
iFluor® 840 succinimidyl ester
iFluor® 560 succinimidyl ester
iFluor® 670 succinimidyl ester
iFluor® 460 succinimidyl ester
iFluor® 440 succinimidyl ester
iFluor® 665 succinimidyl ester
iFluor® 690 succinimidyl ester
iFluor® 720 succinimidyl ester
iFluor® 740 succinimidyl ester
iFluor® 597 succinimidyl ester
iFluor® 770 succinimidyl ester
iFluor® 780 succinimidyl ester
iFluor® 570 succinimidyl ester
iFluor® 830 acid
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Show More (263)

OverviewpdfSDSpdfProtocol


Molecular weight
1436.72
Absorbance (nm)
585
Correction Factor (260 nm)
0.07
Correction Factor (280 nm)
0.05
Extinction coefficient (cm -1 M -1)
1800001
Excitation (nm)
586
Emission (nm)
601
Quantum yield
0.911
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 594 are far superior to the conjugates of other existing similar dyes such as Alexa Fluor® 594. iFluor® Ultra 594 conjugates are significantly brighter than the conjugates prepared with Alexa Fluor® 594 under the same conditions. Additionally, the fluorescence of iFluor® Ultra 594 is not affected by pH (4-10). iFluor® Ultra 594 SE dye is reasonably stable and shows good reactivity and selectivity with protein amino groups. iFluor® Ultra 594 has spectral properties and reactivity similar to Alexa Fluor® 594 (Alexa Fluor® is the trademark of ThermoFisher).

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™ Ultra 594 SE stock solution (Solution B)
Add anhydrous DMSO into the vial of iFluor™ Ultra 594 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™ Ultra 594 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® Ultra 594 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 mM69.603 µL348.015 µL696.03 µL3.48 mL6.96 mL
5 mM13.921 µL69.603 µL139.206 µL696.03 µL1.392 mL
10 mM6.96 µL34.801 µL69.603 µL348.015 µL696.03 µL

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)585
Correction Factor (260 nm)0.07
Correction Factor (280 nm)0.05
Extinction coefficient (cm -1 M -1)1800001
Excitation (nm)586
Emission (nm)601
Quantum yield0.911

Product family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
iFluor® Ultra 647 succinimidyl ester65567025000010.3910.070.07
iFluor® Ultra 750 succinimidyl ester74977325000010.3210.040.05

References


View all 50 references: Citation Explorer
MicroRNA-126 inhibits pathological retinal neovascularization via suppressing vascular endothelial growth factor expression in a rat model of retinopathy of prematurity.
Authors: Fan, Yuan-Yao and Liu, Chi-Hsien and Wu, An-Lun and Chen, Hung-Chi and Hsueh, Yi-Jen and Chen, Kuan-Jen and Lai, Chi-Chun and Huang, Chung-Ying and Wu, Wei-Chi
Journal: European journal of pharmacology (2021): 174035
Retinal ganglion cells projecting to superior colliculus and pulvinar in marmoset.
Authors: Grünert, Ulrike and Lee, Sammy C S and Kwan, William C and Mundinano, Inaki-Carril and Bourne, James A and Martin, Paul R
Journal: Brain structure & function (2021)
A fully integrated isotachophoresis with a programmable microfluidic platform.
Authors: Shebindu, Adam and Somaweera, Himali and Estlack, Zachary and Kim, Jungtae and Kim, Jungkyu
Journal: Talanta (2021): 122039
Liquid Droplet Formation and Facile Cytosolic Translocation of IgG in the Presence of Attenuated Cationic Amphiphilic Lytic Peptides.
Authors: Iwata, Takahiro and Hirose, Hisaaki and Sakamoto, Kentarou and Hirai, Yusuke and Arafiles, Jan Vincent V and Akishiba, Misao and Imanishi, Miki and Futaki, Shiroh
Journal: Angewandte Chemie (International ed. in English) (2021)
Effect of VIRP1 Protein on Nuclear Import of Citrus Exocortis Viroid (CEVd).
Authors: Seo, Hyesu and Kim, Kyunghee and Park, Woong June
Journal: Biomolecules (2021)
Fluorescently-labeled fremanezumab is distributed to sensory and autonomic ganglia and the dura but not to the brain of rats with uncompromised blood brain barrier.
Authors: Noseda, Rodrigo and Schain, Aaron J and Melo-Carrillo, Agustin and Tien, Jason and Stratton, Jennifer and Mai, Fanny and Strassman, Andrew M and Burstein, Rami
Journal: Cephalalgia : an international journal of headache (2020): 229-240
Modeling iontophoretic drug delivery in a microfluidic device.
Authors: Moarefian, Maryam and Davalos, Rafael V and Tafti, Danesh K and Achenie, Luke E and Jones, Caroline N
Journal: Lab on a chip (2020): 3310-3321
Cell-based immunofluorescence assay for screening the neurogenesis potential of new drugs in adult hippocampal neural progenitor cells.
Authors: Zhang, Kun and Li, Bin and Li, Peifang and Yang, Xiaoli and Cui, Huixian and Liu, Xiaoyun
Journal: Acta neurobiologiae experimentalis (2019): 302-308
Super blinking and biocompatible nanoprobes based on dye doped BSA nanoparticles for super resolution imaging.
Authors: Zong, Shenfei and Pan, Fengmei and Zhang, Ruohu and Chen, Chen and Wang, Zhuyuan and Cui, Yiping
Journal: Nanotechnology (2019): 065701
[Sepsis impairs aggregation of nicotinic acetylcholine receptors on murine skeletal muscle cell membranes by inhibiting AKT/GSK3β phosphorylation].
Authors: Li, Tianmei and Liu, Li and Wang, Xiaobin
Journal: Nan fang yi ke da xue xue bao = Journal of Southern Medical University (2019): 1337-1343