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ReadiCleave™ iFluor 546 AML-NHS ester
Fluorescence-based methods have many advantages for biological detections in terms of sensitivity and convenience. Many biological molecules can be readily labeled with a fluorescent tag for fluorescence imaging and flow cytometry analysis. However, most of the existing fluorescent tags are used to permanently labeling biological targets from which the added fluorescent tags cannot be cleaved for further downstream analysis, such as mass spectral analysis or another detection mode. AAT Bioquest’s ReadiCleave™ linkers enable fluorescent tags conjugated to a biological target from which the added fluorescent tag can be removed when needed. ReadiCleave™ iFluor® 546 AML contains an azidomethyl linker that can be cleaved with TCEP to remove the iFluor® 546 fluorophore from the target molecule. The cleavage can be carried out by adding 10-100 mM TCEP solution (pH 7.5) and incubating at 65 °C for 1-5 min. iFluor® 546 is a superior replacement to Alexa Fluor® 546. iFluor® 546 and Alexa Fluor® 546 have very similar spectral properties.
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. ReadiCleave™ iFluor 546 AML-NHS ester stock solution (Solution B)
Add anhydrous DMSO into the vial of ReadiCleave™ iFluor 546 AML-NHS 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 ReadiCleave™ iFluor 546 AML-NHS 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.
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
Product family
| Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Quantum yield | Correction Factor (260 nm) | Correction Factor (280 nm) |
| ReadiCleave™ iFluor 700 AML-NHS ester | 690 | 713 | 2200001 | 0.231 | 0.09 | 0.04 |
| ReadiCleave™ iFluor 488 AML-NHS ester | 491 | 516 | 750001 | 0.91 | 0.21 | 0.11 |
| ReadiCleave™ iFluor 594 AML-NHS ester | 587 | 603 | 2000001 | 0.531 | 0.05 | 0.04 |
References
View all 40 references: Citation Explorer
Evaluation of Blood-Brain Barrier Integrity Using Vascular Permeability Markers: Evans Blue, Sodium Fluorescein, Albumin-Alexa Fluor Conjugates, and Horseradish Peroxidase.
Authors: Ahishali, Bulent and Kaya, Mehmet
Journal: Methods in molecular biology (Clifton, N.J.) (2021): 87-103
Authors: Ahishali, Bulent and Kaya, Mehmet
Journal: Methods in molecular biology (Clifton, N.J.) (2021): 87-103
Effects of Viscosity and Refractive Index on the Emission and Diffusion Properties of Alexa Fluor 405 Using Fluorescence Correlation and Lifetime Spectroscopies.
Authors: van Zanten, Camila and Melnikau, Dzmitry and Ryder, Alan G
Journal: Journal of fluorescence (2021): 835-845
Authors: van Zanten, Camila and Melnikau, Dzmitry and Ryder, Alan G
Journal: Journal of fluorescence (2021): 835-845
Molecular and Spectroscopic Characterization of Green and Red Cyanine Fluorophores from the Alexa Fluor and AF Series*.
Authors: Gebhardt, Christian and Lehmann, Martin and Reif, Maria M and Zacharias, Martin and Gemmecker, Gerd and Cordes, Thorben
Journal: Chemphyschem : a European journal of chemical physics and physical chemistry (2021)
Authors: Gebhardt, Christian and Lehmann, Martin and Reif, Maria M and Zacharias, Martin and Gemmecker, Gerd and Cordes, Thorben
Journal: Chemphyschem : a European journal of chemical physics and physical chemistry (2021)
Hot-Band Anti-Stokes Fluorescence Properties of Alexa Fluor 568.
Authors: Gajdos, Tamás and Hopp, Béla and Erdélyi, Miklós
Journal: Journal of fluorescence (2020): 437-443
Authors: Gajdos, Tamás and Hopp, Béla and Erdélyi, Miklós
Journal: Journal of fluorescence (2020): 437-443
Photo-isomerization of the Cyanine Dye Alexa-Fluor 647 (AF-647) in the Context of dSTORM Super-Resolution Microscopy.
Authors: Karlsson, Joshua K G and Laude, Alex and Hall, Michael J and Harriman, Anthony
Journal: Chemistry (Weinheim an der Bergstrasse, Germany) (2019): 14983-14998
Authors: Karlsson, Joshua K G and Laude, Alex and Hall, Michael J and Harriman, Anthony
Journal: Chemistry (Weinheim an der Bergstrasse, Germany) (2019): 14983-14998
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