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Amplite® Fluorimetric Tyrosinase Assay Kit

Tyrosinase dose response was measured with Amplite® Fluorimetric Tyrosinase Assay Kit in a 96-well black plate using a Gemini microplate reader (Molecular Devices). Equal volume of Tyrosinase standards and Tyrosinase Blue were added and incubated for 6 hours at 37 °C.  The signal was acquired at Ex/Em = 340/440 nm (cut off at 420 nm).
Tyrosinase dose response was measured with Amplite® Fluorimetric Tyrosinase Assay Kit in a 96-well black plate using a Gemini microplate reader (Molecular Devices). Equal volume of Tyrosinase standards and Tyrosinase Blue were added and incubated for 6 hours at 37 °C.  The signal was acquired at Ex/Em = 340/440 nm (cut off at 420 nm).
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Catalog Number11312
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OverviewpdfSDSpdfProtocol


Tyrosinase is an enzyme expressed across a vast range of species from bacteria and fungi to animals. 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® Fluorimetric 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 fluorogenic substrate that significantly increases its fluorescence intensity at 440 nm upon reaction with tyrosinase. The increases in fluorescence intensity at 440 nm is well correlated with tyrosinase activity. The assay kit is designed to be run with a fluorescence microplate reader.

Platform


Fluorescence microplate reader

Excitation340 nm
Emission440 nm
Cutoff420 nm
Recommended plateSolid black

Components


Component A: Tyrosinase Standard1 vial
Component B: Assay Buffer1 bottle (20 mL)
Component C: Tyrosinase Blue1 vial
Component D: DMSO1 vial (100 µL)

Example protocol


AT A GLANCE

Protocol Summary
  1. Prepare and add standards and samples (50 µL)
  2. Prepare and add Tyrosinase Blue working solution to the standards and samples wells (50 µL)
  3. Incubate the plate at room temperature for 60 to 120 minutes
  4. Monitor the fluorescence intensity at Ex/Em= 340/440 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 a single use aliquots.


2. Tyrosinase Blue stock solution (100X):
Add 50 µL DMSO (Component D) into Tyrosinase Blue (Component C) and mix well. Note: Store the unused Tyrosinase Blue 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/11312


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 concentrations of the standards in wells will be 2X. Note: With provided standards, 2 standard curves can be generated in duplicates if using at suggested concentrations.

PREPARATION OF WORKING SOLUTION

Tyrosinase Blue working solution
Make a 1:100 dilution by adding 5 µL Tyrosinase Blue stock solution (100X) to 1 mL Assay Buffer (Component B) and mix well.

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
T1 T1 TS TS
T2 T2    
T3 T3    
T4 T4    
T5 T5    
T6 T6    
T7 T7    
BL BL    
  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 Blue working solution to the wells of standards and samples.
  3. Incubate the reaction at 37 °C for 60 to 120 minutes.
    Note      The reaction can be kept up to 6 hours.
  4. Monitor the fluorescence intensity with fluorescence plate reader at Ex/Em= 340/440 nm with cutoff= 420 nm. 

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