iFluor® 665 maleimide
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, 405, 488, 532, 555, 633, and 647 nm). The iFluor® 665 family has spectral properties similar to those of Alexa Fluor® 660 (Alexa Fluor® is the trademark of Invitrogen). In addition, the fluorescence of iFluor® 665 is pH-insensitive over a broad range, pH 3-11. These spectral characteristics make this new dye family an excellent alternative to Alexa Fluor® 660. Under the same conditions, iFluor® 665 gives a stronger fluorescence signal on some antibodies we tested. iFluor® 665 maleimide is reasonably stable and shows good reactivity and selectivity with protein thiol groups even under neutral or slightly acidic conditions.
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 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 upto 4 weeks when kept from light and moisture. Avoid freeze-thaw cycles.
Note The pH of the protein solution (Solution A) should be 6.5 ± 0.5.
Note Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or other proteins will not be labeled well.
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.
Optional: if your protein does not contain a free cysteine, you must treat your protein with DTT or TCEP to generate a thiol group. DTT or TCEP are used for converting a disulfide bond to two free thiol groups. If DTT is used you must remove free DTT by dialysis or gel filtration before conjugating a dye maleimide to your protein. Following is a sample protocol for generating a free thiol group:
1. iFluor™ 665 maleimide stock solution (Solution B)
Add anhydrous DMSO into the vial of iFluor™ 665 maleimide 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 upto 4 weeks when kept from light and moisture. Avoid freeze-thaw cycles.
2. Protein stock solution (Solution A)
Mix 100 µL of a reaction buffer (e.g., 100 mM MES buffer with pH ~6.0) 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 6.5 ± 0.5.
Note Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or other proteins will not be labeled well.
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.
Optional: if your protein does not contain a free cysteine, you must treat your protein with DTT or TCEP to generate a thiol group. DTT or TCEP are used for converting a disulfide bond to two free thiol groups. If DTT is used you must remove free DTT by dialysis or gel filtration before conjugating a dye maleimide to your protein. Following is a sample protocol for generating a free thiol group:
- Prepare a fresh solution of 1 M DTT (15.4 mg/100 µL) in distilled water.
- Make IgG solution in 20 mM DTT: add 20 µL of DTT stock per ml of IgG solution while mixing. Let stand at room temp for 30 minutes without additional mixing (to minimize reoxidation of cysteines to cystines).
- Pass the reduced IgG over a filtration column pre-equilibrated with "Exchange Buffer". Collect 0.25 mL fractions off the column.
- Determine the protein concentrations and pool the fractions with the majority of the IgG. This can be done either spectrophotometrically or colorimetrically.
- Carry out the conjugation as soon as possible after this step (see Sample Experiment Protocol).
Note IgG solutions should be >4 mg/mL for the best results. The antibody should be concentrated if less than 2 mg/mL. Include an extra 10% for losses on the buffer exchange column.
Note The reduction can be carried out in almost any buffers from pH 7-7.5, e.g., MES, phosphate or TRIS buffers.
Note Steps 3 and 4 can be replaced by dialysis.
SAMPLE EXPERIMENTAL PROTOCOL
This labeling protocol was developed for the conjugate of Goat anti-mouse IgG with iFluor™ 665 maleimide. 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.
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 maleimide | 345 | 450 | 200001 | 0.951 | 0.83 | 0.23 |
iFluor® 488 maleimide | 491 | 516 | 750001 | 0.91 | 0.21 | 0.11 |
iFluor® 555 maleimide | 557 | 570 | 1000001 | 0.641 | 0.23 | 0.14 |
iFluor® 647 maleimide | 656 | 670 | 2500001 | 0.251 | 0.03 | 0.03 |
iFluor® 680 maleimide | 684 | 701 | 2200001 | 0.231 | 0.097 | 0.094 |
iFluor® 700 maleimide | 690 | 713 | 2200001 | 0.231 | 0.09 | 0.04 |
iFluor® 750 maleimide | 757 | 779 | 2750001 | 0.121 | 0.044 | 0.039 |
iFluor® 790 maleimide | 787 | 812 | 2500001 | 0.131 | 0.1 | 0.09 |
iFluor® 800 maleimide | 801 | 820 | 2500001 | 0.111 | 0.03 | 0.08 |
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References
View all 7 references: Citation Explorer
Recognition of Invasive Prostate Cancer Using a GHRL Polypeptide Probe Targeting GHSR in a Mouse Model In Vivo.
Authors: Ye, Huamao and Yang, Yue and Chen, Rui and Shi, Xiaolei and Fang, Yu and Yang, Jun and Dong, Yuanzhen and Chen, Lili and Xia, Jianghua and Wang, Chao and Yang, Chenghua and Feng, Jun and Wang, Yang and Feng, Xiang and Lü, Chen
Journal: Current pharmaceutical design (2020): 1614-1621
Authors: Ye, Huamao and Yang, Yue and Chen, Rui and Shi, Xiaolei and Fang, Yu and Yang, Jun and Dong, Yuanzhen and Chen, Lili and Xia, Jianghua and Wang, Chao and Yang, Chenghua and Feng, Jun and Wang, Yang and Feng, Xiang and Lü, Chen
Journal: Current pharmaceutical design (2020): 1614-1621
A rapid sensitive, flow cytometry-based method for the detection of Plasmodium vivax-infected blood cells.
Authors: Roobsoong, Wanlapa and Maher, Steven P and Rachaphaew, Nattawan and Barnes, Samantha J and Williamson, Kim C and Sattabongkot, Jetsumon and Adams, John H
Journal: Malaria journal (2014): 55
Authors: Roobsoong, Wanlapa and Maher, Steven P and Rachaphaew, Nattawan and Barnes, Samantha J and Williamson, Kim C and Sattabongkot, Jetsumon and Adams, John H
Journal: Malaria journal (2014): 55
Reengineering the optical absorption cross-section of photosynthetic reaction centers.
Authors: Dutta, Palash K and Lin, Su and Loskutov, Andrey and Levenberg, Symon and Jun, Daniel and Saer, Rafael and Beatty, J Thomas and Liu, Yan and Yan, Hao and Woodbury, Neal W
Journal: Journal of the American Chemical Society (2014): 4599-604
Authors: Dutta, Palash K and Lin, Su and Loskutov, Andrey and Levenberg, Symon and Jun, Daniel and Saer, Rafael and Beatty, J Thomas and Liu, Yan and Yan, Hao and Woodbury, Neal W
Journal: Journal of the American Chemical Society (2014): 4599-604
Comparison of a chimeric anti-carcinoembryonic antigen antibody conjugated with visible or near-infrared fluorescent dyes for imaging pancreatic cancer in orthotopic nude mouse models.
Authors: Maawy, Ali A and Hiroshima, Yukihiko and Kaushal, Sharmeela and Luiken, George A and Hoffman, Robert M and Bouvet, Michael
Journal: Journal of biomedical optics (2013): 126016
Authors: Maawy, Ali A and Hiroshima, Yukihiko and Kaushal, Sharmeela and Luiken, George A and Hoffman, Robert M and Bouvet, Michael
Journal: Journal of biomedical optics (2013): 126016
Nucleic acid sandwich hybridization assay with quantum dot-induced fluorescence resonance energy transfer for pathogen detection.
Authors: Chou, Cheng-Chung and Huang, Yi-Han
Journal: Sensors (Basel, Switzerland) (2012): 16660-72
Authors: Chou, Cheng-Chung and Huang, Yi-Han
Journal: Sensors (Basel, Switzerland) (2012): 16660-72
Page updated on October 8, 2024