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Amplite® Colorimetric Tyrosinase Assay Kit
Tyrosinase is of great interest to drug discovery, life science research, food industry and cosmetics industry since it plays an important role in the biosynthetic pathway of melanin. The development and screening of tyrosinase inhibitors has received great attentions to melanoma related illnesses. Tyrosinase levels and activity are highly upregulated in melanoma and considered to a reliable test to monitor melanoma related illnesses. AAT Bioquest has developed Amplite® Colorimetric Tyrosinase Assay Kit. It is a simple, one-step and reliable assay for monitoring tyrosinase activity with very high sensitivity. The assay uses a proprietary substrate colorless solution that significantly increases its absorption at 510 nm upon reaction with tyrosinase. The increases in absorption at 510 nm is well correlated with tyrosinase activity. The assay kit is designed to be run with a microplate reader.
Example protocol
AT A GLANCE
Protocol Summary
- Prepare and add standards and samples (50 µL)
- Prepare and add Tyrosinase Substrate working solution to the standards and samples wells (50 µL)
- Incubate the plate at room temperature for 30 to 60 minutes
- Monitor the absorbance at 510 nm
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 Store the unused Tyrosinase stock solution at -20 °C in single use aliquots.
Note Store the unused Tyrosinase Substrate stock solution at -20 °C in single use aliquots.
1. Tyrosinase stock solution (2000 U/mL)
Add 120 µL Assay Buffer (Component B) into Tyrosinase Standard (Component A) and mix well. Note Store the unused Tyrosinase stock solution at -20 °C in single use aliquots.
2. Tyrosinase Substrate stock solution (50X)
Add 100 µL ddH2O into Assay Substrate (Component C) and mix well. Note Store the unused Tyrosinase Substrate stock solution at -20 °C in single use aliquots.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/11311
https://www.aatbio.com/tools/serial-dilution/11311
Tyrosinase standard
Use Tyrosinase stock solution (2000 U/mL) and Assay Buffer to generate 400 U/mL final concentration of Tyrosinase Standard solution (T1). Then perform 1:2 serial dilutions to get remaining serially diluted Tyrosinase Standards (T2-T7). Note: The final in well concentration of the standards will be 2X. Note: With provided standard, 2 standard curves can be performed in duplicates if using at suggested concentrations.PREPARATION OF WORKING SOLUTION
Tyrosinase Substrate working solution
Make a 1:50 dilution by adding 20 µL Tyrosinase Substrate stock solution (50X) and 20 µL Tyrosinase Enhancer (Component D) into 1 mL Assay Buffer (Component B) and mix well. Note Tyrosinase Substrate working solution should be made immediately upon use. We recommend using working solution within several hours.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1.Layout of Tyrosinase standards and test samples in a white-clear bottom 96- wells microplate. Tyrosinase standards (T1-T7= 400 to 6.25 U/mL), TS= Test Samples, BL= Blank samples
The following protocol can be used as a guideline and should be optimized according to the needs.
| T1 | T1 | TS | TS |
| T2 | T2 | ||
| T3 | T3 | ||
| T4 | T4 | ||
| T5 | T5 | ||
| T6 | T6 | ||
| T7 | T7 | ||
| BL | BL |
- Prepare the standards and test samples as per recommendations in assay buffer and add 50 µL of each in a microplate.
- Add 50 µL Tyrosinase Substrate working solution to the wells of standards and samples.
- Incubate the reaction at room temperature for 30 to 60 minutes.
- Monitor the absorbance with an absorbance plate reader at 510 nm.
Citations
View all 10 citations: Citation Explorer
A comprehensive review on tyrosinase inhibitors
Authors: Zolghadri, S., Bahrami, A., Hassan Khan, M. T., Munoz-Munoz, J., Garcia-Molina, F., Garcia-Canovas, F., Saboury, A. A.
Journal: J Enzyme Inhib Med Chem (2019): 279-309
Authors: Zolghadri, S., Bahrami, A., Hassan Khan, M. T., Munoz-Munoz, J., Garcia-Molina, F., Garcia-Canovas, F., Saboury, A. A.
Journal: J Enzyme Inhib Med Chem (2019): 279-309
Tyrosinase (TYR) gene sequencing and literature review reveals recurrent mutations and multiple population founder gene mutations as causative of oculocutaneous albinism (OCA) in Pakistani families
Authors: Shakil, M., Harlalka, G. V., Ali, S., Lin, S., D'Atri, I., Hussain, S., Nasir, A., Shahzad, M. A., Ullah, M. I., Self, J. E., Baple, E. L., Crosby, A. H., Mahmood, S.
Journal: Eye (Lond) (2019): ersion="1.0" encoding="UTF-8" ?>11311.enlEndN
Authors: Shakil, M., Harlalka, G. V., Ali, S., Lin, S., D'Atri, I., Hussain, S., Nasir, A., Shahzad, M. A., Ullah, M. I., Self, J. E., Baple, E. L., Crosby, A. H., Mahmood, S.
Journal: Eye (Lond) (2019): ersion="1.0" encoding="UTF-8" ?>11311.enlEndN
Moraceae Plants with Tyrosinase Inhibitory Activity: A Review
Authors: Burl, undefined and o, B., Clericuzio, M., Cornara, L.
Journal: Mini Rev Med Chem (2017): 108-121
Authors: Burl, undefined and o, B., Clericuzio, M., Cornara, L.
Journal: Mini Rev Med Chem (2017): 108-121
Tyrosinase inhibitors: a patent review (2011-2015)
Authors: Ullah, S., Son, S., Yun, H. Y., Kim, D. H., Chun, P., Moon, H. R.
Journal: Expert Opin Ther Pat (2016): 347-62
Authors: Ullah, S., Son, S., Yun, H. Y., Kim, D. H., Chun, P., Moon, H. R.
Journal: Expert Opin Ther Pat (2016): 347-62
Critical review of Ayurvedic Varnya herbs and their tyrosinase inhibition effect
Authors: Sharma, K., Joshi, N., Goyal, C.
Journal: Anc Sci Life (2015): 18-25
Authors: Sharma, K., Joshi, N., Goyal, C.
Journal: Anc Sci Life (2015): 18-25
Page updated on November 21, 2024
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