iFluor® 488 succinimidyl ester
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Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
Quotation | Request |
International | See distributors |
Shipping | Standard overnight for United States, inquire for international |
Molecular weight | 945.07 |
Solvent | DMSO |
Correction Factor (260 nm) | 0.21 |
Correction Factor (280 nm) | 0.11 |
Extinction coefficient (cm -1 M -1) | 750001 |
Excitation (nm) | 491 |
Emission (nm) | 516 |
Quantum yield | 0.91 |
Certificate of Origin | Download PDF |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
Storage | Freeze (< -15 °C); Minimize light exposure |
UNSPSC | 12171501 |
Overview | ![]() ![]() |
Molecular weight 945.07 | Correction Factor (260 nm) 0.21 | Correction Factor (280 nm) 0.11 | Extinction coefficient (cm -1 M -1) 750001 | Excitation (nm) 491 | Emission (nm) 516 | Quantum yield 0.91 |
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
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. The final protein concentration range of 2-10 mg/mL is recommended for optimal labeling efficiency.
Add anhydrous DMSO into the vial of iFluor™ 488 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 the 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™ 488 SE. You might need further optimization for your particular proteins.
Note: Each protein requires a distinct dye/protein ratio, which also depends on the properties of dyes. Over-labeling of a protein could detrimentally affect its binding affinity, while the protein conjugates of low dye/protein ratio give reduced sensitivity.
Use a 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 using a 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.
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 must be diluted with staining buffer, and aliquoted for multiple uses.
Note: For longer-term storage, the dye-protein conjugate solution needs to be concentrated or freeze-dried.
Calculators
Common stock solution preparation
0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
1 mM | 105.812 µL | 529.061 µL | 1.058 mL | 5.291 mL | 10.581 mL |
5 mM | 21.162 µL | 105.812 µL | 211.625 µL | 1.058 mL | 2.116 mL |
10 mM | 10.581 µL | 52.906 µL | 105.812 µL | 529.061 µL | 1.058 mL |
Molarity calculator
Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
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Spectrum

Spectral properties
Correction Factor (260 nm) | 0.21 |
Correction Factor (280 nm) | 0.11 |
Extinction coefficient (cm -1 M -1) | 750001 |
Excitation (nm) | 491 |
Emission (nm) | 516 |
Quantum yield | 0.91 |
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® 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 | 588 | 604 | 1800001 | 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 |
iFluor® 660 succinimidyl ester | 663 | 678 | 2500001 | 0.261 | 0.07 | 0.08 |
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Citations
Authors: Constant, David A and Gutierrez, Jahir M and Sastry, Anand V and Viazzo, Rebecca and Smith, Nicholas R and Hossain, Jubair and Spencer, David A and Carter, Hayley and Ventura, Abigail B and Louie, Michael TM and others,
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Authors: Teran, Oriana Y and Zanotelli, Matthew R and Lin, Miao-chong Joy and Cerione, Richard A and Wilson, Kristin F
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Journal: (2023)
Authors: Kellaway, Sophie G and Potluri, Sandeep and Keane, Peter and Blair, Helen and Chin, Paulynn S and Ptasinska, Anetta and Worker, Alice and Ames, Luke and Adamo, Assunta and Coleman, Daniel JL and others,
Journal: bioRxiv (2023): 2023--03
Authors: Wang, Jingyu and Xu, Lintao and Peng, Deqing and Zhu, Yongjian and Gu, Zhaowen and Yao, Ying and Li, Heyangzi and Cao, Xi and Fu, Chun-yan and Zheng, Mingzhi and others,
Journal: Inflammation and Regeneration (2023): 1--15
Authors: Colombo, Federico and Villiou, Maria and Taheri, Fereydoon and Fr{\"o}hlich, Leonard and Taale, Mohammadreza and Albert, Viktoria and Jiang, Qiyang and Selhuber-Unkel, Christine
Journal: Advanced NanoBiomed Research (2023): 2200156
Authors: Wang, Dan and Qu, Hao and Kang, Hui and Xu, Feng and Huang, Wei and Cai, Xianhua
Journal: Bioengineered (2022): 8772--8784
Authors: Popovici, Violeta and Matei, Elena and Cozaru, Georgeta Camelia and Bucur, Laura and G{\^\i}rd, Cerasela Elena and Schr{\"o}der, Verginica and Ozon, Emma Adriana and Karampelas, Oana and Musuc, Adina Magdalena and Atkinson, Irina and others,
Journal: Antioxidants (2022): 1601
Authors: Yang, Chih-Hsiang and Fagnocchi, Luca and Apostle, Stefanos and Wegert, Vanessa and Casan{\'\i}-Gald{\'o}n, Salvador and Landgraf, Kathrin and Panzeri, Ilaria and Dror, Erez and Heyne, Steffen and W{\"o}rpel, Till and others,
Journal: Nature metabolism (2022): 1150--1165
Authors: Zhu, Xinyu and Trimarco, Joseph D and Williams, Courtney A and Barrera, Alejandro and Reddy, Timothy E and Heaton, Nicholas S
Journal: Cell Reports (2022): 111540
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