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iFluor® 665 succinimidyl ester
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, 532, 555, 633, and 647 nm). The iFluor® 665 family has spectral properties similar to those of Alexa Fluor® 660 (Alexa Fluor® is the trademark of Invitrogen). In addition, the fluorescence of iFluor® 665 is pH-insensitive over a broad range, pH 3-11. These spectral characteristics make this new dye family an excellent alternative to Alexa Fluor® 660. Under the same conditions, iFluor® 665 gives a stronger fluorescence signal on some antibodies we tested. iFluor® 665 SE is reasonably stable and shows good reactivity and selectivity with protein amino groups.
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™ 665 SE stock solution (Solution B)
Add anhydrous DMSO into the vial of iFluor™ 665 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™ 665 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® 350 succinimidyl ester | 345 | 450 | 200001 | 0.951 | 0.83 | 0.23 |
| iFluor® 405 succinimidyl ester | 403 | 427 | 370001 | 0.911 | 0.48 | 0.77 |
| iFluor® 488 succinimidyl ester | 491 | 516 | 750001 | 0.91 | 0.21 | 0.11 |
| iFluor® 514 succinimidyl ester | 511 | 527 | 750001 | 0.831 | 0.265 | 0.116 |
| iFluor® 532 succinimidyl ester | 537 | 560 | 900001 | 0.681 | 0.26 | 0.16 |
| iFluor® 555 succinimidyl ester | 557 | 570 | 1000001 | 0.641 | 0.23 | 0.14 |
| iFluor® 594 succinimidyl ester | 587 | 603 | 2000001 | 0.531 | 0.05 | 0.04 |
| iFluor® 633 succinimidyl ester | 640 | 654 | 2500001 | 0.291 | 0.062 | 0.044 |
| iFluor® 647 succinimidyl ester | 656 | 670 | 2500001 | 0.251 | 0.03 | 0.03 |
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References
View all 7 references: Citation Explorer
Recognition of Invasive Prostate Cancer Using a GHRL Polypeptide Probe Targeting GHSR in a Mouse Model In Vivo.
Authors: Ye, Huamao and Yang, Yue and Chen, Rui and Shi, Xiaolei and Fang, Yu and Yang, Jun and Dong, Yuanzhen and Chen, Lili and Xia, Jianghua and Wang, Chao and Yang, Chenghua and Feng, Jun and Wang, Yang and Feng, Xiang and Lü, Chen
Journal: Current pharmaceutical design (2020): 1614-1621
Authors: Ye, Huamao and Yang, Yue and Chen, Rui and Shi, Xiaolei and Fang, Yu and Yang, Jun and Dong, Yuanzhen and Chen, Lili and Xia, Jianghua and Wang, Chao and Yang, Chenghua and Feng, Jun and Wang, Yang and Feng, Xiang and Lü, Chen
Journal: Current pharmaceutical design (2020): 1614-1621
A rapid sensitive, flow cytometry-based method for the detection of Plasmodium vivax-infected blood cells.
Authors: Roobsoong, Wanlapa and Maher, Steven P and Rachaphaew, Nattawan and Barnes, Samantha J and Williamson, Kim C and Sattabongkot, Jetsumon and Adams, John H
Journal: Malaria journal (2014): 55
Authors: Roobsoong, Wanlapa and Maher, Steven P and Rachaphaew, Nattawan and Barnes, Samantha J and Williamson, Kim C and Sattabongkot, Jetsumon and Adams, John H
Journal: Malaria journal (2014): 55
Reengineering the optical absorption cross-section of photosynthetic reaction centers.
Authors: Dutta, Palash K and Lin, Su and Loskutov, Andrey and Levenberg, Symon and Jun, Daniel and Saer, Rafael and Beatty, J Thomas and Liu, Yan and Yan, Hao and Woodbury, Neal W
Journal: Journal of the American Chemical Society (2014): 4599-604
Authors: Dutta, Palash K and Lin, Su and Loskutov, Andrey and Levenberg, Symon and Jun, Daniel and Saer, Rafael and Beatty, J Thomas and Liu, Yan and Yan, Hao and Woodbury, Neal W
Journal: Journal of the American Chemical Society (2014): 4599-604
Comparison of a chimeric anti-carcinoembryonic antigen antibody conjugated with visible or near-infrared fluorescent dyes for imaging pancreatic cancer in orthotopic nude mouse models.
Authors: Maawy, Ali A and Hiroshima, Yukihiko and Kaushal, Sharmeela and Luiken, George A and Hoffman, Robert M and Bouvet, Michael
Journal: Journal of biomedical optics (2013): 126016
Authors: Maawy, Ali A and Hiroshima, Yukihiko and Kaushal, Sharmeela and Luiken, George A and Hoffman, Robert M and Bouvet, Michael
Journal: Journal of biomedical optics (2013): 126016
Nucleic acid sandwich hybridization assay with quantum dot-induced fluorescence resonance energy transfer for pathogen detection.
Authors: Chou, Cheng-Chung and Huang, Yi-Han
Journal: Sensors (Basel, Switzerland) (2012): 16660-72
Authors: Chou, Cheng-Chung and Huang, Yi-Han
Journal: Sensors (Basel, Switzerland) (2012): 16660-72
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