Amplite® Fluorimetric Tyrosinase Assay Kit

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Intended use

Research Use Only (RUO)

Overview SDS Protocol

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

Excitation	340 nm
Emission	440 nm
Cutoff	420 nm
Recommended plate	Solid black

Components

Example protocol

AT A GLANCE

Protocol Summary

Prepare and add standards and samples (50 µL)
Prepare and add Tyrosinase Blue working solution to the standards and samples wells (50 µL)
Incubate the plate at room temperature for 60 to 120 minutes
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

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

Images

Figure 1. 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).

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