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N-Me-DMAE, NHS ester
The acridinium NHS ester can be used to label proteins and nucleic acids. The covalently bound acridinium NHS esters generate chemiluminescence in the presence of hydrogen peroxide. Exposure of an acridinium ester label to an alkaline hydrogen peroxide solution triggers a flash of light. Acridinium-labeled proteins and other biological molecules can be used as sensitive detection methods in immunoassays and other biological detections. N-Me-DMAE acridinium probe can be readily used for labeling proteins (such as antibodies) and other amino-containing biomolecules. Compared to Lumiwox™ acridinium NHS ester (#26000), the acridinium label is released from the carrier (such as proteins) upon hydrogen peroxide activation. If you need the acridinium ester label remains bonded with the carrier, you would need use Lumiwox™ acridinium NHS ester instead.
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. N-Me-DMAE, NHS ester stock solution (Solution B)
Add anhydrous DMSO into the vial of N-Me-DMAE, 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 N-Me-DMAE, 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.
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 N-Me-DMAE, NHS 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 | 158.088 µL | 790.439 µL | 1.581 mL | 7.904 mL | 15.809 mL |
| 5 mM | 31.618 µL | 158.088 µL | 316.176 µL | 1.581 mL | 3.162 mL |
| 10 mM | 15.809 µL | 79.044 µL | 158.088 µL | 790.439 µL | 1.581 mL |
Molarity calculator
Enter any two values (mass, volume, concentration) to calculate the third.
| Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
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Citations
View all 27 citations: Citation Explorer
On the use of acridinium indicators for the chemiluminescent determination of the total antioxidant capacity of dietary supplements
Authors: Krzyminski, K. K., Roshal, A. D., Rudnicki-Velasquez, P. B., Zamojc, K.
Journal: Luminescence (2019): ersion="1.0" encoding="UTF-8" ?>26005.enlEndN
Authors: Krzyminski, K. K., Roshal, A. D., Rudnicki-Velasquez, P. B., Zamojc, K.
Journal: Luminescence (2019): ersion="1.0" encoding="UTF-8" ?>26005.enlEndN
A novel chemiluminescent immunoassay based on original acridinium ester labels as better solution for diagnosis of human toxoplasmosis than conventional ELISA test
Authors: Holec-Gasior, L., Ferra, B., Czechowska, J., Serdiuk, I. E., Krzyminski, K.
Journal: Diagn Microbiol Infect Dis (2018): 13-19
Authors: Holec-Gasior, L., Ferra, B., Czechowska, J., Serdiuk, I. E., Krzyminski, K.
Journal: Diagn Microbiol Infect Dis (2018): 13-19
Enhancement effect on the chemiluminescence of acridinium esters under neutral conditions
Authors: Nakazono, M., Nanbu, S.
Journal: Luminescence (2018): 345-348
Authors: Nakazono, M., Nanbu, S.
Journal: Luminescence (2018): 345-348
A comparison of chemiluminescent acridinium dimethylphenyl ester labels with different conjugation sites
Authors: Natrajan, A., Wen, D.
Journal: Org Biomol Chem (2015): 2622-33
Authors: Natrajan, A., Wen, D.
Journal: Org Biomol Chem (2015): 2622-33
Chemiluminescence accompanied by the reaction of acridinium ester and manganese (II)
Authors: Ren, L., Cui, H.
Journal: Luminescence (2014): 929-32
Authors: Ren, L., Cui, H.
Journal: Luminescence (2014): 929-32
Page updated on November 23, 2024
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