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iFluor® Ultra 647 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 647 are far superior to the conjugates of other existing similar dyes such as Cy5, Dylight 650 and Alexa Fluor® 647. iFluor® Ultra 647 conjugates are significantly brighter than the conjugates prepared with Cy5, Dylight 650 and Alexa Fluor® 647 under the same conditions. Additionally, the fluorescence of iFluor® Ultra 647 is not affected by pH (4-10). iFluor® Ultra 647 SE dye is reasonably stable and shows good reactivity and selectivity with protein amino groups. iFluor® Ultra 647 has spectral properties and reactivity similar to Cy5, Dylight 650 and Alexa Fluor® 647 (Cy5® and Alexa Fluor® is the trademarks of GE Healthcare and ThermoFisher respectively).
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.
Note Once reconstituted, the NHS ester reactive dye solution is not very stable, especially if exposed to moisture. It could hydrolyze into the nonreactive free acid in aqueous solutions.
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 ~8.5) 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 647 SE stock solution (Solution B)
Add 100 µL high-quality, anhydrous dimethylsulfoxide (DMSO) or dimethyl-formamide (DMF) to 1 mg iFluor™ Ultra 647 SE to prepare 10 mg/mL 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.
Note Once reconstituted, the NHS ester reactive dye solution is not very stable, especially if exposed to moisture. It could hydrolyze into the nonreactive free acid in aqueous solutions.
SAMPLE EXPERIMENTAL PROTOCOL
This labeling protocol was developed for the conjugate of Goat anti-mouse IgG with iFluor™ Ultra 647 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 6-10 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.
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 with 15 mL MWCO=30K filter (https://www.emdmillipore.com/US/en/product/Amicon-Ultra-4-Centrifugal-Filter-Units,MM_NF-C7719)- Prepare column according to the manufacture instruction.
- Load the reaction mixture (From "Run conjugation reaction") to the top of the column.
- Add 4 mL of PBS (pH 7.2-7.4) as soon as the sample runs.
- Centrifuge to concentrate to ~0.4 mL.
- Add 4 mL PBS and then concentrate to ~0.4 mL.
- Repeat ~3 times, until the elution absorbance at 650 nm < 0.1.
- Collect the purified Antibody-iFluor™Ultra 647 conjugate solution.
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 488 succinimidyl ester | 491 | 516 | 750001 | 0.91 | 0.21 | 0.11 |
| iFluor® Ultra 594 succinimidyl ester | 586 | 600 | 1800001 | 0.521 | 0.07 | 0.05 |
| iFluor® Ultra 750 succinimidyl ester | 749 | 773 | 2500001 | 0.321 | 0.04 | 0.05 |
Citations
View all 5 citations: Citation Explorer
Site-specific labeling and functional efficiencies of human fibroblast growth Factor-1 with a range of fluorescent Dyes in the flexible N-Terminal region and a rigid $\beta$-turn region
Authors: Mohale, Mamello and Gundampati, Ravi Kumar and Kumar, Thallapuranam Krishnaswamy Suresh and Heyes, Colin D
Journal: Analytical biochemistry (2022): 114524
Authors: Mohale, Mamello and Gundampati, Ravi Kumar and Kumar, Thallapuranam Krishnaswamy Suresh and Heyes, Colin D
Journal: Analytical biochemistry (2022): 114524
SP/NK-1R Axis Promotes Perineural Invasion of Pancreatic Cancer and is Affected by lncRNA LOC389641
Authors: Ji, Tengfei and Ma, Keqiang and Wu, Hongsheng and Cao, Tiansheng
Journal: (2021)
Authors: Ji, Tengfei and Ma, Keqiang and Wu, Hongsheng and Cao, Tiansheng
Journal: (2021)
Pharmacological targeting of Sam68 functions in colorectal cancer stem cells
Authors: Masibag, Angelique N and Bergin, Christopher J and Haebe, Joshua R and Zouggar, A{\"\i}cha and Shah, Muhammad S and Sandouka, Tamara and da Silva, Amanda Mendes and Desrochers, Fran{\c{c}}ois M and Fournier-Morin, Aube and Benoit, Yannick D
Journal: Iscience (2021): 103442
Authors: Masibag, Angelique N and Bergin, Christopher J and Haebe, Joshua R and Zouggar, A{\"\i}cha and Shah, Muhammad S and Sandouka, Tamara and da Silva, Amanda Mendes and Desrochers, Fran{\c{c}}ois M and Fournier-Morin, Aube and Benoit, Yannick D
Journal: Iscience (2021): 103442
Efferocytosis induces macrophage proliferation to help resolve tissue injury
Authors: Gerlach, Brennan D and Ampomah, Patrick B and Yurdagul Jr, Arif and Liu, Chuang and Lauring, Max C and Wang, Xiaobo and Kasikara, Canan and Kong, Na and Shi, Jinjun and Tao, Wei and others,
Journal: Cell metabolism (2021): 2445--2463
Authors: Gerlach, Brennan D and Ampomah, Patrick B and Yurdagul Jr, Arif and Liu, Chuang and Lauring, Max C and Wang, Xiaobo and Kasikara, Canan and Kong, Na and Shi, Jinjun and Tao, Wei and others,
Journal: Cell metabolism (2021): 2445--2463
Influence of particle geometry on gastrointestinal transit and absorption following oral administration
Authors: Li, Dong and Zhuang, Jie and He, Haisheng and Jiang, Sifan and Banerjee, Amrita and Lu, Yi and Wu, Wei and Mitragotri, Samir and Gan, Li and Qi, Jianping
Journal: ACS applied materials \& interfaces (2017): 42492--42502
Authors: Li, Dong and Zhuang, Jie and He, Haisheng and Jiang, Sifan and Banerjee, Amrita and Lu, Yi and Wu, Wei and Mitragotri, Samir and Gan, Li and Qi, Jianping
Journal: ACS applied materials \& interfaces (2017): 42492--42502
References
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Journal: Advanced therapeutics (2021)
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Journal: Advanced therapeutics (2021)
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Molecular and Spectroscopic Characterization of Green and Red Cyanine Fluorophores from the Alexa Fluor and AF Series*.
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