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Amplite® Colorimetric Tyrosinase Assay Kit

Tyrosinase dose response was measured with Amplite® Colorimetric Tyrosinase Assay Kit in a 96-well white plate using a SpectraMax microplate reader (Molecular Devices). Equal volume of Tyrosinase standards and Tyrosinase Substrate with enhancer were added and incubated for 30 minutes at room temperature.  The signal was acquired at 510 nm.
Tyrosinase dose response was measured with Amplite® Colorimetric Tyrosinase Assay Kit in a 96-well white plate using a SpectraMax microplate reader (Molecular Devices). Equal volume of Tyrosinase standards and Tyrosinase Substrate with enhancer were added and incubated for 30 minutes at room temperature.  The signal was acquired at 510 nm.
Tyrosinase dose response was measured with Amplite® Colorimetric Tyrosinase Assay Kit in a 96-well white plate using a SpectraMax microplate reader (Molecular Devices). Equal volume of Tyrosinase standards and Tyrosinase Substrate with enhancer were added and incubated for 30 minutes at room temperature.  The signal was acquired at 510 nm.
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OverviewpdfSDSpdfProtocol


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.

Platform


Absorbance microplate reader

Absorbance510 nm
Recommended plateWhite plate/Clear bottom

Components


Example protocol


AT A GLANCE

Protocol Summary
  1. Prepare and add standards and samples (50 µL)
  2. Prepare and add Tyrosinase Substrate working solution to the standards and samples wells (50 µL)
  3. Incubate the plate at room temperature for 30 to 60 minutes
  4. Monitor the absorbance at 510 nm 
Important      Bring all the kit components at room temperature before starting the experiment.

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. 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


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
T1T1TSTS
T2T2  
T3T3  
T4T4  
T5T5  
T6T6  
T7T7  
BLBL  
The following protocol can be used as a guideline and should be optimized according to the needs.
  1. Prepare the standards and test samples as per recommendations in assay buffer and add 50 µL of each in a microplate.
  2. Add 50 µL Tyrosinase Substrate working solution to the wells of standards and samples.
  3. Incubate the reaction at room temperature for 30 to 60 minutes.
  4. Monitor the absorbance with an absorbance plate reader at 510 nm. 

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Citations


View all 10 citations: Citation Explorer
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
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
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
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
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
A short review on the structure-function relationship of artificial catecholase/tyrosinase and nuclease activities of Cu-complexes
Authors: Silavi, R., Divsalar, A., Saboury, A. A.
Journal: J Biomol Struct Dyn (2012): 752-72
An updated review of tyrosinase inhibitors
Authors: Chang, T. S.
Journal: Int J Mol Sci (2009): 2440-75
A review on spectrophotometric methods for measuring the monophenolase and diphenolase activities of tyrosinase
Authors: Garcia-Molina, F., Munoz, J. L., Varon, R., Rodriguez-Lopez, J. N., Garcia-Canovas, F., Tudela, J.
Journal: J Agric Food Chem (2007): 9739-49
Clinical significance of sequential tyrosinase expression in the peripheral blood of disease-free melanoma patients: a review of literature data
Authors: Quaglino, P., Savoia, P., Fierro, M. T., Osella-Abate, S., Bernengo, M. G.
Journal: Melanoma Res (2004): S17-9
Tyrosinase: a comprehensive review of its mechanism
Authors: Sanchez-Ferrer, A., Rodriguez-Lopez, J. N., Garcia-Canovas, F., Garcia-Carmona, F.
Journal: Biochim Biophys Acta (1995): 1-11