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iFluor® 594 succinimidyl ester
AAT Bioquest's iFluor® dyes are optimized for labeling proteins, particularly antibodies. iFluor® 594 dyes have fluorescence excitation and emission maxima of ~588 nm and ~604 nm respectively. iFluor® 594 family has spectral properties similar to those of Texas Red® and Alexa Fluor® 594 (Texas Red® and Alexa Fluor® 594 are the trademarks of Invitrogen). iFluor® 594 family is pH-independent from pH 3 to 11. These spectral characteristics make this new dye family an excellent alternative to Texas Red® and Alexa Fluor® 594. Compared to Texas Red®, iFluor® 594 is much easier to conjugate with RPE with a much higher conjugation yield, and the resulting RPE-iFluor® 594 tandem has better FRET efficiency. iFluor® 594 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™ 594 SE stock solution (Solution B)
Add anhydrous DMSO into the vial of iFluor™ 594 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™ 594 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.
Calculators
Common stock solution preparation
Table 1. Volume of DMSO needed to reconstitute specific mass of iFluor® 594 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 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
| 1 mM | 86.176 µL | 430.878 µL | 861.757 µL | 4.309 mL | 8.618 mL |
| 5 mM | 17.235 µL | 86.176 µL | 172.351 µL | 861.757 µL | 1.724 mL |
| 10 mM | 8.618 µL | 43.088 µL | 86.176 µL | 430.878 µL | 861.757 µL |
Molarity calculator
Enter any two values (mass, volume, concentration) to calculate the third.
| Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
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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® 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 |
Show More (36) | ||||||
Citations
View all 9 citations: Citation Explorer
A broadly applicable protein-polymer adjuvant system for antiviral vaccines
Authors: Wang, Caiqian and Geng, Yuanyuan and Wang, Haoran and Ren, Zeheng and Hou, Qingxiu and Fang, An and Wu, Qiong and Wu, Liqin and Shi, Xiujuan and Zhou, Ming and others,
Journal: EMBO Molecular Medicine (2024): 1--33
Authors: Wang, Caiqian and Geng, Yuanyuan and Wang, Haoran and Ren, Zeheng and Hou, Qingxiu and Fang, An and Wu, Qiong and Wu, Liqin and Shi, Xiujuan and Zhou, Ming and others,
Journal: EMBO Molecular Medicine (2024): 1--33
Endurance exercise upregulates mtp expression in aged Drosophila to ameliorate age-related diastolic dysfunction and extend lifespan
Authors: Peng, Tianhang and Ding, Meng and Yan, Hanhui and Zhang, Ping and Tian, Rui and Guo, Yin and Zheng, Lan
Journal: Physiological Reports (2024): e15929
Authors: Peng, Tianhang and Ding, Meng and Yan, Hanhui and Zhang, Ping and Tian, Rui and Guo, Yin and Zheng, Lan
Journal: Physiological Reports (2024): e15929
Oncolytic adenovirus H101 enhanced antitumor effects of PD-1 blockade by downregulating CD47 on tumor cells
Authors: Qiao, Chenxiao and Wang, Song and Xu, Yipeng and He, Yedie and Cai, Zhijian and Wang, Hua
Journal: (2023)
Authors: Qiao, Chenxiao and Wang, Song and Xu, Yipeng and He, Yedie and Cai, Zhijian and Wang, Hua
Journal: (2023)
Multiplex Immunofluorescence: A Powerful Tool in Cancer Immunotherapy
Authors: Sheng, Wenjie and Zhang, Chaoyu and Mohiuddin, TM and Al-Rawe, Marwah and Zeppernick, Felix and Falcone, Franco H and Meinhold-Heerlein, Ivo and Hussain, Ahmad Fawzi
Journal: International Journal of Molecular Sciences (2023): 3086
Authors: Sheng, Wenjie and Zhang, Chaoyu and Mohiuddin, TM and Al-Rawe, Marwah and Zeppernick, Felix and Falcone, Franco H and Meinhold-Heerlein, Ivo and Hussain, Ahmad Fawzi
Journal: International Journal of Molecular Sciences (2023): 3086
Targeted Micellar Phthalocyanine for Lymph Node Metastasis Homing and Photothermal Therapy in an Orthotopic Colorectal Tumor Model
Authors: Feng, Hai-Yi and Yuan, Yihang and Zhang, Yunpeng and Liu, Hai-Jun and Dong, Xiao and Yang, Si-Cong and Liu, Xue-Liang and Lai, Xing and Zhu, Mao-Hua and Wang, Jue and others,
Journal: Nano-Micro Letters (2021): 1--17
Authors: Feng, Hai-Yi and Yuan, Yihang and Zhang, Yunpeng and Liu, Hai-Jun and Dong, Xiao and Yang, Si-Cong and Liu, Xue-Liang and Lai, Xing and Zhu, Mao-Hua and Wang, Jue and others,
Journal: Nano-Micro Letters (2021): 1--17
References
View all 49 references: Citation Explorer
Sequential ordering among multicolor fluorophores for protein labeling facility via aggregation-elimination based beta-lactam probes
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Journal: Mol Biosyst (2011): 1766
Authors: Sadhu KK, Mizukami S, Watanabe S, Kikuchi K.
Journal: Mol Biosyst (2011): 1766
Visualizing dengue virus through Alexa Fluor labeling
Authors: Zhang S, Tan HC, Ooi EE.
Journal: J Vis Exp. (2011)
Authors: Zhang S, Tan HC, Ooi EE.
Journal: J Vis Exp. (2011)
Fluorescent "Turn-on" system utilizing a quencher-conjugated peptide for specific protein labeling of living cells
Authors: Arai S, Yoon SI, Murata A, Takabayashi M, Wu X, Lu Y, Takeoka S, Ozaki M.
Journal: Biochem Biophys Res Commun (2011): 211
Authors: Arai S, Yoon SI, Murata A, Takabayashi M, Wu X, Lu Y, Takeoka S, Ozaki M.
Journal: Biochem Biophys Res Commun (2011): 211
Neuroanatomical basis of clinical joint application of "Jinggu" (BL 64, a source-acupoint) and "Dazhong" (KI 4, a Luo-acupoint) in the rat: a double-labeling study of cholera toxin subunit B conjugated with Alexa Fluor 488 and 594
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Journal: Zhen Ci Yan Jiu (2011): 262
Authors: Cui JJ, Zhu XL, Ji CF, Jing XH, Bai WZ.
Journal: Zhen Ci Yan Jiu (2011): 262
Simultaneous detection of virulence factors from a colony in diarrheagenic Escherichia coli by a multiplex PCR assay with Alexa Fluor-labeled primers
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