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iFluor® 740 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, 355, 405, 488, 555, 633, 638, 647, 660, and 802 nm). iFluor® 740 dyes have fluorescence excitation and emission maxima of ~742 nm and ~764 nm respectively. These spectral characteristics make them a unique color for fluorescence imaging and flow cytometry applications. iFluor® 740 is an excellent acceptor dye for preparing tandem colors with APC and PE. These iFluor® 740 tandem colors offer a set of unique color profiles for spectral flow cytometry. Compared to Alexa Fluor 700 tandems, iFluor® 740 tandems have improved photostability.
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™ 740 SE stock solution (Solution B)
Add anhydrous DMSO into the vial of iFluor™ 740 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™ 740 SE. You might need further optimization for your particular proteins. 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 8 references: Citation Explorer
Multiplexed non-invasive tumor imaging of glucose metabolism and receptor-ligand engagement using dark quencher FRET acceptor.
Authors: Rudkouskaya, Alena and Sinsuebphon, Nattawut and Ochoa, Marien and Chen, Sez-Jade and Mazurkiewicz, Joseph E and Intes, Xavier and Barroso, Margarida
Journal: Theranostics (2020): 10309-10325
Authors: Rudkouskaya, Alena and Sinsuebphon, Nattawut and Ochoa, Marien and Chen, Sez-Jade and Mazurkiewicz, Joseph E and Intes, Xavier and Barroso, Margarida
Journal: Theranostics (2020): 10309-10325
Performance of optoacoustic and fluorescence imaging in detecting deep-seated fluorescent agents.
Authors: Chen, Zhenyue and Deán-Ben, Xosé Luís and Gottschalk, Sven and Razansky, Daniel
Journal: Biomedical optics express (2018): 2229-2239
Authors: Chen, Zhenyue and Deán-Ben, Xosé Luís and Gottschalk, Sven and Razansky, Daniel
Journal: Biomedical optics express (2018): 2229-2239
An enzymatically-sensitized sequential and concentric energy transfer relay self-assembled around semiconductor quantum dots.
Authors: Samanta, Anirban and Walper, Scott A and Susumu, Kimihiro and Dwyer, Chris L and Medintz, Igor L
Journal: Nanoscale (2015): 7603-14
Authors: Samanta, Anirban and Walper, Scott A and Susumu, Kimihiro and Dwyer, Chris L and Medintz, Igor L
Journal: Nanoscale (2015): 7603-14
Multicolor detection of rare tumor cells in blood using a novel flow cytometry-based system.
Authors: Watanabe, Masaru and Uehara, Yuri and Yamashita, Namiko and Fujimura, Yuu and Nishio, Kaori and Sawada, Takeshi and Takeda, Kazuo and Koizumi, Fumiaki and Koh, Yasuhiro
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2014): 206-13
Authors: Watanabe, Masaru and Uehara, Yuri and Yamashita, Namiko and Fujimura, Yuu and Nishio, Kaori and Sawada, Takeshi and Takeda, Kazuo and Koizumi, Fumiaki and Koh, Yasuhiro
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2014): 206-13
Noninvasive and quantitative assessment of in vivo fetomaternal interface angiogenesis using RGD-based fluorescence.
Authors: Keramidas, M and Lavaud, J and Sergent, F and Hoffmann, P and Brouillet, S and Feige, J-J and Coll, J-L and Alfaidy, N
Journal: BioMed research international (2014): 309082
Authors: Keramidas, M and Lavaud, J and Sergent, F and Hoffmann, P and Brouillet, S and Feige, J-J and Coll, J-L and Alfaidy, N
Journal: BioMed research international (2014): 309082
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