iFluor® 750 succinimidyl ester
AAT Bioquest's iFluor® dyes are developed for labeling proteins, particularly antibodies. These dyes are optimized to have minimal fluorescence quenching effect on proteins and nucleic acids. Our iFluor® 750 dyes have fluorescence excitation and emission maxima of ~757 nm and ~779 nm, respectively, with good photostability. Our in-house comparable studies indicated that our iFluor® 750 dyes are significantly brighter than the corresponding Cy7® and Alexa Fluor® 750. These spectral characteristics make them a superior alternative to Cy7® and Alexa Fluor® 750 (Cy7® and Alexa Fluor® are the trademarks of GE Healthcare and Invitrogen). iFluor® 750 conjugates have been widely used in fluorescence animal imaging applications. The iFluor® 750 SE dye is reasonably stable and shows good reactivity and selectivity with proteins.
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™ 750 SE stock solution (Solution B)
Add anhydrous DMSO into the vial of iFluor™ 750 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™ 750 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® 750 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 | 70.58 µL | 352.9 µL | 705.801 µL | 3.529 mL | 7.058 mL |
5 mM | 14.116 µL | 70.58 µL | 141.16 µL | 705.801 µL | 1.412 mL |
10 mM | 7.058 µL | 35.29 µL | 70.58 µL | 352.9 µL | 705.801 µL |
Molarity calculator
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Spectrum
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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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Citations
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In Vivo Visualization of Collagen Transdermal Absorption by a Combined Second-harmonic Generation and Two-photon Excited Fluorescence Method
Authors: Ma, Shuhua and He, Gaiying and Yang, Weifeng and Wang, Yi and others,
Journal: (2021)
Authors: Ma, Shuhua and He, Gaiying and Yang, Weifeng and Wang, Yi and others,
Journal: (2021)
Deep Sequencing Analysis of the Eha-Regulated Transcriptome of Edwardsiella tarda Following Acidification
Authors: Gao, D and Liu, N and Li, Y and Zhang, Y and Liu, G and others, undefined
Journal: Metabolomics (Los Angel) (2017): 2153--0769
Authors: Gao, D and Liu, N and Li, Y and Zhang, Y and Liu, G and others, undefined
Journal: Metabolomics (Los Angel) (2017): 2153--0769
Suramin inhibits cullin-RING E3 ubiquitin ligases
Authors: Wu, Kenneth and Chong, Robert A and Yu, Qing and Bai, Jin and Spratt, Donald E and Ching, Kevin and Lee, Chan and Miao, Haibin and Tappin, Inger and Hurwitz, Jerard and others, undefined
Journal: Proceedings of the National Academy of Sciences (2016): E2011--E2018
Authors: Wu, Kenneth and Chong, Robert A and Yu, Qing and Bai, Jin and Spratt, Donald E and Ching, Kevin and Lee, Chan and Miao, Haibin and Tappin, Inger and Hurwitz, Jerard and others, undefined
Journal: Proceedings of the National Academy of Sciences (2016): E2011--E2018
Glycosaminoglycan mimicry by COAM reduces melanoma growth through chemokine induction and function
Authors: Piccard, Helene and Berghmans, Nele and Korpos, Eva and Dillen, Chris and Aelst, Ilse Van and Li, S and ra , undefined and Martens, Erik and Liekens, S and ra , undefined and Noppen, Sam and Damme, Jo Van and others, undefined
Journal: International Journal of Cancer (2012): E425--E436
Authors: Piccard, Helene and Berghmans, Nele and Korpos, Eva and Dillen, Chris and Aelst, Ilse Van and Li, S and ra , undefined and Martens, Erik and Liekens, S and ra , undefined and Noppen, Sam and Damme, Jo Van and others, undefined
Journal: International Journal of Cancer (2012): E425--E436
References
View all 49 references: Citation Explorer
Sequential ordering among multicolor fluorophores for protein labeling facility via aggregation-elimination based beta-lactam probes
Authors: Sadhu KK, Mizukami S, Watanabe S, Kikuchi K.
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Authors: Sadhu KK, Mizukami S, Watanabe S, Kikuchi K.
Journal: Mol Biosyst (2011): 1766
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Journal: J Vis Exp. (2011)
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Journal: J Vis Exp. (2011)
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Authors: Arai S, Yoon SI, Murata A, Takabayashi M, Wu X, Lu Y, Takeoka S, Ozaki M.
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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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Simultaneous detection of virulence factors from a colony in diarrheagenic Escherichia coli by a multiplex PCR assay with Alexa Fluor-labeled primers
Authors: Kuwayama M, Shigemoto N, Oohara S, Tanizawa Y, Yamada H, Takeda Y, Matsuo T, Fukuda S.
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Journal: J Microbiol Methods (2011): 119
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