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iFluor® Ultra 750 succinimidyl ester
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.
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.
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.
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.
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.
Spectrum
Product family
| Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Quantum yield | Correction Factor (260 nm) | Correction Factor (280 nm) |
| iFluor® Ultra 594 succinimidyl ester | 586 | 600 | 1800001 | 0.521 | 0.07 | 0.05 |
| iFluor® Ultra 647 succinimidyl ester | 655 | 670 | 2500001 | 0.391 | 0.07 | 0.07 |
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)
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
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
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
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
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
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