Cal-520® NHS Ester
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Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
Molecular weight | 1048.9 |
Solvent | DMSO |
Excitation (nm) | 492 |
Emission (nm) | 515 |
Quantum yield | 0.751 |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
Storage | Freeze (< -15 °C); Minimize light exposure |
UNSPSC | 12352200 |
Overview | ![]() ![]() |
Molecular weight 1048.9 | Excitation (nm) 492 | Emission (nm) 515 | Quantum yield 0.751 |
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
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. The final protein concentration range of 2-10 mg/mL is recommended for optimal labeling efficiency.
Add anhydrous DMSO into the vial of Cal-520® 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 the 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 Cal-520® NHS ester. You might need further optimization for your particular proteins.
Note: Each protein requires a distinct dye/protein ratio, which also depends on the properties of dyes. Over-labeling of a protein could detrimentally affect its binding affinity, while the protein conjugates of low dye/protein ratio give reduced sensitivity.
Use a 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 using a 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.
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 must be diluted with staining buffer, and aliquoted for multiple uses.
Note: For longer-term storage, the dye-protein conjugate solution needs to be concentrated or freeze-dried.
Calculators
Common stock solution preparation
0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
1 mM | 95.338 µL | 476.69 µL | 953.38 µL | 4.767 mL | 9.534 mL |
5 mM | 19.068 µL | 95.338 µL | 190.676 µL | 953.38 µL | 1.907 mL |
10 mM | 9.534 µL | 47.669 µL | 95.338 µL | 476.69 µL | 953.38 µL |
Molarity calculator
Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
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Spectrum
![spectrum](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fspectra%2Fcal_520.png&w=2048&q=50)
Spectral properties
Excitation (nm) | 492 |
Emission (nm) | 515 |
Quantum yield | 0.751 |
Citations
Authors: Schultz, Simon R and Copel, undefined and , Caroline S and Foust, Am and a J , undefined and Quicke, Peter and Schuck, Renaud
Journal: Proceedings of the IEEE (2017): 139--157
Authors: Daily, Neil J and Du, Zhong-Wei and Wakatsuki, Tetsuro
Journal: ASSAY and Drug Development Technologies (2017)
Authors: Kettenhofen, Ralf
Journal: Stem Cell-Derived Models in Toxicology (2017): 135--152
Authors: Servin-Vences, M Rocio and Moroni, Mirko and Lewin, Gary R and Poole, Kate
Journal: eLife (2017): e21074
Authors: Sun, Wei and He, Shihua and Martínez-Romero, Carles and Kouznetsova, Jennifer and Tawa, Gregory and Xu, Miao and Shinn, Paul and Fisher, Ethan G and Long, Yan and Motabar, Omid and others, undefined
Journal: Antiviral Research (2017): 165--172
Authors: Mitsui, Retsu and Hashitani, Hikaru
Journal: Pflügers Archiv-European Journal of Physiology (2017): 1--14
Authors: Pan, Libiao and Yang, Junhua and Yang, Qian and Wang, Xiaomeng and Zhu, Liya and Liu, Yali and Lou, Huifang and Xu, Chou and Shen, Ying and Wang, Hao
Journal: Frontiers in molecular neuroscience (2017)
Authors: Shi, Xuefeng and Barchini, Jad and Ledesma, Hector Acaron and Koren, David and Jin, Yanjiao and Liu, Xiaorong and Wei, Wei and Cang, Jianhua
Journal: Nature Neuroscience (2017)
Authors: Kodama, Daisuke and Hirai, Takao and Kondo, Hisataka and Hamamura, Kazunori and Togari, Akifumi
Journal: FEBS Letters (2017)
Authors: Daily, Neil J and Santos, Radleigh and Vecchi, Joseph and Kemanli, Pinar and Wakatsuki, Tetsuro
Journal: Journal of evolving stem cell research (2017): 1
Application notes
Abbreviation of Common Chemical Compounds Related to Peptides
Bright Tide Fluor™-Based Fluorescent Peptides and Their Applications In Drug Discovery and Disease Diagnosis
FITC (Fluorescein isothiocyanate)
Fluorescein isothiocyanate (FITC)