iFluor® 770 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® 770 dyes have fluorescence excitation and emission maxima of ~777 nm and ~797 nm respectively. These spectral characteristics make them a unique color for fluorescence imaging and flow cytometry applications. iFluor® 770 is an excellent acceptor dye for preparing tandem colors with APC and PE. These iFluor® 770 tandem colors offer a set of unique color profiles for spectral flow cytometry. iFluor® 770 is one of the brightest NIR dyes, and some of its antibody conjugates are significantly brighter than those prepared with IRDyes of similar wavelengths, such as IRDye 800RS. iFluor® 770 succinimidyl ester is an amine-reactive form used to conjugate with amine-containing molecules such as antibodies and peptides.
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™ 770 SE stock solution (Solution B)
Add anhydrous DMSO into the vial of iFluor™ 770 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™ 770 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
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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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References
View all 50 references: Citation Explorer
Molecular imaging of a fluorescent antibody against epidermal growth factor receptor detects high-grade glioma.
Authors: Zhou, Quan and Vega Leonel, Johana C M and Santoso, Michelle Rai and Wilson, Christy and van den Berg, Nynke S and Chan, Carmel T and Aryal, Muna and Vogel, Hannes and Cayrol, Romain and Mandella, Michael J and Schonig, Frank and Lu, Guolan and Gambhir, Sanjiv S and Moseley, Michael E and Rosenthal, Eben L and Grant, Gerald A
Journal: Scientific reports (2021): 5710
Authors: Zhou, Quan and Vega Leonel, Johana C M and Santoso, Michelle Rai and Wilson, Christy and van den Berg, Nynke S and Chan, Carmel T and Aryal, Muna and Vogel, Hannes and Cayrol, Romain and Mandella, Michael J and Schonig, Frank and Lu, Guolan and Gambhir, Sanjiv S and Moseley, Michael E and Rosenthal, Eben L and Grant, Gerald A
Journal: Scientific reports (2021): 5710
Comparison of HER2-Targeted Antibodies for Fluorescence-Guided Surgery in Breast Cancer.
Authors: AghaAmiri, Solmaz and Simien, Jo and Thompson, Alastair M and Voss, Julie and Ghosh, Sukhen C and Hernandez Vargas, Servando and Kim, Sarah and Azhdarinia, Ali and Tran Cao, Hop S
Journal: Molecular imaging (2021): 5540569
Authors: AghaAmiri, Solmaz and Simien, Jo and Thompson, Alastair M and Voss, Julie and Ghosh, Sukhen C and Hernandez Vargas, Servando and Kim, Sarah and Azhdarinia, Ali and Tran Cao, Hop S
Journal: Molecular imaging (2021): 5540569
Predicting Schwannoma Growth in a Tumor Model Using Targeted Imaging.
Authors: Morrison, Daniel R and Sorace, Anna G and Hamilton, Ellis and Moore, Lindsay S and Houson, Hailey A and Udayakumar, Neha and Ovaitt, Alyssa and Warram, Jason M and Walsh, Erika M
Journal: Otology & neurotology : official publication of the American Otological Society, American Neurotology Society [and] European Aca (2021): e615-e623
Authors: Morrison, Daniel R and Sorace, Anna G and Hamilton, Ellis and Moore, Lindsay S and Houson, Hailey A and Udayakumar, Neha and Ovaitt, Alyssa and Warram, Jason M and Walsh, Erika M
Journal: Otology & neurotology : official publication of the American Otological Society, American Neurotology Society [and] European Aca (2021): e615-e623
Resection and survival data from a clinical trial of glioblastoma multiforme-specific IRDye800-BBN fluorescence-guided surgery.
Authors: He, Kunshan and Chi, Chongwei and Li, Deling and Zhang, Jingjing and Niu, Gang and Lv, Fangqiao and Wang, Junmei and Che, Wenqiang and Zhang, Liwei and Ji, Nan and Zhu, Zhaohui and Tian, Jie and Chen, Xiaoyuan
Journal: Bioengineering & translational medicine (2021): e10182
Authors: He, Kunshan and Chi, Chongwei and Li, Deling and Zhang, Jingjing and Niu, Gang and Lv, Fangqiao and Wang, Junmei and Che, Wenqiang and Zhang, Liwei and Ji, Nan and Zhu, Zhaohui and Tian, Jie and Chen, Xiaoyuan
Journal: Bioengineering & translational medicine (2021): e10182
Preclinical Development of Near-Infrared-Labeled CD38-Targeted Daratumumab for Optical Imaging of CD38 in Multiple Myeloma.
Authors: Cho, Nicholas and Ko, Sooah and Shokeen, Monica
Journal: Molecular imaging and biology (2021): 186-195
Authors: Cho, Nicholas and Ko, Sooah and Shokeen, Monica
Journal: Molecular imaging and biology (2021): 186-195
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