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zFluor™ 647 succinimidyl ester
Our zFluor™ serial dyes are developed to have the possibly highest photostability at a given wavelength compared to the other similar wavelength dyes on the market. zFluor™ 647 has almost identical spectral properties to the popular Cy5® (GE Healthcare) and Alexa Fluor® 647 (ThermoFisher). Its high thermal and photostability makes it an excellent choice for single-molecule detection applications and high-resolution microscopy such as PALM, dSTORM and STED. Under the same test conditions, zFluor™ 647 has much higher ozone stability than Cy5® and Alexa Fluor® 647, making it a much better choice for microarray and other biochip-based applications. In addition, zFluor™-labeled oligonucleotides and peptides are much brighter and more photostable than the ones labeled by Alexa Fluor® 647 and Cy5®. This feature is extremely useful for fluorescence in-situ hybridization (FISH). In common with our iFluor® labels, the absorption and fluorescence of our zFluor™ 647 are independent of pH in the range of pH 2 to 11. It is well excited at 633 nm of He-Ne laser, the 647 nm line of the Krypton-Ion laser or a diode-laser emitting at 650 nm.
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.
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. zFluor™ 647 succinimidyl ester stock solution (Solution B)
Add anhydrous DMSO into the vial of zFluor™ 647 succinimidyl ester 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 zFluor™ 647 succinimidyl ester. 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.
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 zFluor™ 647 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 | 117.228 µL | 586.139 µL | 1.172 mL | 5.861 mL | 11.723 mL |
| 5 mM | 23.446 µL | 117.228 µL | 234.456 µL | 1.172 mL | 2.345 mL |
| 10 mM | 11.723 µL | 58.614 µL | 117.228 µL | 586.139 µL | 1.172 mL |
Molarity calculator
Enter any two values (mass, volume, concentration) to calculate the third.
| Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
| / | = | x | = |
Spectrum
Product family
| Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Quantum yield | Correction Factor (260 nm) | Correction Factor (280 nm) |
| zFluor™ 633 succinimidyl ester | 623 | 639 | - | - | - | - |
| zFluor™ 635 succinimidyl ester | - | - | 110,0001 | 0.221 | 0.12 | 0.09 |
| iFluor® 647 succinimidyl ester | 656 | 670 | 2500001 | 0.251 | 0.03 | 0.03 |
References
View all 19 references: Citation Explorer
Single nucleotide polymorphism discrimination with and without an ethidium bromide intercalator
Authors: Fenati, R. A.; Connolly, A. R.; Ellis, A. V.
Journal: Anal Chim Acta (2017): 121-128
Authors: Fenati, R. A.; Connolly, A. R.; Ellis, A. V.
Journal: Anal Chim Acta (2017): 121-128
Generation and Characterization of Virus-Enhancing Peptide Nanofibrils Functionalized with Fluorescent Labels
Authors: Rode, S.; Hayn, M.; Rocker, A.; Sieste, S.; Lamla, M.; Markx, D.; Meier, C.; Kirchhoff, F.; Walther, P.; F and rich, M.; Weil, T.; Munch, J.
Journal: Bioconjug Chem (2017): 1260-1270
Authors: Rode, S.; Hayn, M.; Rocker, A.; Sieste, S.; Lamla, M.; Markx, D.; Meier, C.; Kirchhoff, F.; Walther, P.; F and rich, M.; Weil, T.; Munch, J.
Journal: Bioconjug Chem (2017): 1260-1270
Capillary-Driven Microfluidic Chips for Miniaturized Immunoassays: Patterning Capture Antibodies Using Microcontact Printing and Dry-Film Resists
Authors: Temiz, Y.; Lovchik, R. D.; Delamarche, E.
Journal: Methods Mol Biol (2017): 37-47
Authors: Temiz, Y.; Lovchik, R. D.; Delamarche, E.
Journal: Methods Mol Biol (2017): 37-47
Background Suppression in Imaging Gold Nanorods through Detection of Anti-Stokes Emission
Authors: Carattino, A.; Keizer, V. I.; Schaaf, M. J.; Orrit, M.
Journal: Biophys J (2016): 2492-2499
Authors: Carattino, A.; Keizer, V. I.; Schaaf, M. J.; Orrit, M.
Journal: Biophys J (2016): 2492-2499
Absolute two-photon excitation spectra of red and far-red fluorescent probes
Authors: Velasco, M. G.; Allgeyer, E. S.; Yuan, P.; Grutzendler, J.; Bewersdorf, J.
Journal: Opt Lett (2015): 4915-8
Authors: Velasco, M. G.; Allgeyer, E. S.; Yuan, P.; Grutzendler, J.; Bewersdorf, J.
Journal: Opt Lett (2015): 4915-8
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