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iFluor® Ultra 750 succinimidyl ester

HeLa cells were incubated with mouse anti-tubulin followed by AAT’s iFluor<sup>TM</sup> Ultra 750 goat anti-mouse IgG conjugate or Alexa Fluor<sup>®</sup> 750 goat anti-mouse IgG.
HeLa cells were incubated with mouse anti-tubulin followed by AAT’s iFluor<sup>TM</sup> Ultra 750 goat anti-mouse IgG conjugate or Alexa Fluor<sup>®</sup> 750 goat anti-mouse IgG.
Ordering information
Price ()
Catalog Number71680
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 weight1426.78
SolventDMSO
Spectral properties
Absorbance (nm)750
Correction Factor (260 nm)0.04
Correction Factor (280 nm)0.05
Extinction coefficient (cm -1 M -1)2500001
Excitation (nm)749
Emission (nm)773
Quantum yield0.321
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® 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*
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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
iFluor 750™ RGD Conjugate
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iFluor® 405 hydrazide
iFluor® 800 acid
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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*
iFluor® 555 Tyramide
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iFluor® 810 goat anti-mouse IgG (H+L)
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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® 840 maleimide
iFluor® 770 maleimide
iFluor® 780 maleimide
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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
1426.78
Absorbance (nm)
750
Correction Factor (260 nm)
0.04
Correction Factor (280 nm)
0.05
Extinction coefficient (cm -1 M -1)
2500001
Excitation (nm)
749
Emission (nm)
773
Quantum yield
0.321
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 750 are far superior to the conjugates of other existing similar dyes such as Cy7, Dylight 755, IRDye750 and Alexa Fluor® 750. iFluor® Ultra 750 conjugates might be the brightest among the NIR dye antibody conjugates. They are significantly brighter than the conjugates prepared with Cy7, Dylight 755, IRDye750 and Alexa Fluor® 750 under the same conditions. Additionally, the fluorescence of iFluor® Ultra 750 is not affected by pH (4-10). iFluor® Ultra 750 SE dye is reasonably stable and shows good reactivity and selectivity with protein amino groups. iFluor® Ultra 750 has spectral properties and reactivity similar to Cy7, Dylight 755, IRDye750 and Alexa Fluor® 750.

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 750 SE stock solution (Solution B)
Add anhydrous DMSO into the vial of iFluor™ Ultra 750 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 750 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 750 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 mM70.088 µL350.439 µL700.879 µL3.504 mL7.009 mL
5 mM14.018 µL70.088 µL140.176 µL700.879 µL1.402 mL
10 mM7.009 µL35.044 µL70.088 µL350.439 µL700.879 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


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spectrum

Spectral properties

Absorbance (nm)750
Correction Factor (260 nm)0.04
Correction Factor (280 nm)0.05
Extinction coefficient (cm -1 M -1)2500001
Excitation (nm)749
Emission (nm)773
Quantum yield0.321

Product family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
iFluor® Ultra 594 succinimidyl ester58660118000010.9110.070.05
iFluor® Ultra 647 succinimidyl ester65567025000010.3910.070.07

References


View all 28 references: Citation Explorer
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)
CD24-targeted intraoperative fluorescence image-guided surgery leads to improved cytoreduction of ovarian cancer in a preclinical orthotopic surgical model.
Authors: Kleinmanns, Katrin and Fosse, Vibeke and Davidson, Ben and de Jalón, Elvira García and Tenstad, Olav and Bjørge, Line and McCormack, Emmet
Journal: EBioMedicine (2020): 102783
Generation and characterization of novel recombinant anti-hERG1 scFv antibodies for cancer molecular imaging.
Authors: Duranti, Claudia and Carraresi, Laura and Sette, Angelica and Stefanini, Matteo and Lottini, Tiziano and Crescioli, Silvia and Crociani, Olivia and Iamele, Luisa and De Jonge, Hugo and Gherardi, Ermanno and Arcangeli, Annarosa
Journal: Oncotarget (2018): 34972-34989
Enhanced Release of Molecules upon Ultraviolet (UV) Light Irradiation from Photoresponsive Hydrogels Prepared from Bifunctional Azobenzene and Four-Arm Poly(ethylene glycol).
Authors: Rastogi, Shiva K and Anderson, Hailee E and Lamas, Joseph and Barret, Scott and Cantu, Travis and Zauscher, Stefan and Brittain, William J and Betancourt, Tania
Journal: ACS applied materials & interfaces (2018): 30071-30080
Phosphorothioate-Modified AP613-1 Specifically Targets GPC3 when Used for Hepatocellular Carcinoma Cell Imaging.
Authors: Dong, Lili and Zhou, Hongxin and Zhao, Menglong and Gao, Xinghui and Liu, Yang and Liu, Dongli and Guo, Wei and Hu, Hongwei and Xie, Qian and Fan, Jia and Lin, Jiang and Wu, Weizhong
Journal: Molecular therapy. Nucleic acids (2018): 376-386
In vivo fluorescence imaging of hepatocellular carcinoma using a novel GPC3-specific aptamer probe.
Authors: Zhao, Menglong and Dong, Lili and Liu, Zhuang and Yang, Shuohui and Wu, Weizhong and Lin, Jiang
Journal: Quantitative imaging in medicine and surgery (2018): 151-160
Performance of optoacoustic and fluorescence imaging in detecting deep-seated fluorescent agents.
Authors: Chen, Zhenyue and Deán-Ben, Xosé Luís and Gottschalk, Sven and Razansky, Daniel
Journal: Biomedical optics express (2018): 2229-2239
Doxorubicin-loaded protease-activated near-infrared fluorescent polymeric nanoparticles for imaging and therapy of cancer.
Authors: Yildiz, Tugba and Gu, Renpeng and Zauscher, Stefan and Betancourt, Tania
Journal: International journal of nanomedicine (2018): 6961-6986
Optimization of magnetic retention in the gastrointestinal tract: Enhanced bioavailability of poorly permeable drug.
Authors: Seth, Anjali and Lafargue, David and Poirier, Cécile and Badier, Thomas and Delory, Nathalie and Laporte, Alain and Delbos, Jean-Marie and Jeannin, Véronique and Péan, Jean-Manuel and Ménager, Christine
Journal: European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences (2017): 25-35
Preclinical evaluation of near-infrared (NIR) fluorescently labeled cetuximab as a potential tool for fluorescence-guided surgery.
Authors: Saccomano, Mara and Dullin, Christian and Alves, Frauke and Napp, Joanna
Journal: International journal of cancer (2016): 2277-89