FOR RESEARCH USE ONLY!
Tyrosinase Inhibitor Screening Kit (Colorimetric)
(Catalog # K575-100; 100 assays; Store at -20°C) 9/14
I. Introduction:
Tyrosinase or polyphenol oxidase (EC [Link]), is an oxidoreductase that participates in the biosynthesis of melanin, a ubiquitous biological
pigment found in hair, eyes, skin, etc. Inhibition of tyrosinase has been a long-time target in the skin health research, cosmetics and
agricultural industries because of its role in browning reactions in skin pigmentation and during fruit harvesting and handling. Skin whitening
and bleaching products utilize natural or synthetic tyrosinase inhibitors in order to lighten the skin color. Polyphenols, benzaldehyde
derivatives, long-chain lipids, steroids, and natural compounds have been used as tyrosinase inhibitors. Tyrosinase catalyzes the oxidation of
tyrosine, producing a chromophore that can be detected at 510 nm. In the presence of Kojic Acid, a reversible inhibitor of tyrosinase, the rate
of oxidation of the substrate is decreased. The kit provides a rapid, simple, sensitive, and reliable test suitable for high-throughput screening
of tyrosinase inhibitors. The assay is also adaptable to a 384-well format.
II. Application:
Screening/characterizing tyrosinase inhibitors
III. Kit Contents:
Components K575-100 Cap Code Part Number
Tyrosinase Assay Buffer 25 ml WM K575-100-1
Tyrosinase Substrate 1 vial Red K575-100-2
Tyrosinase (Lyophilized) 1 vial Blue K575-100-3
Tyrosinase Enhancer 0.5 ml Purple K575-100-4
Inhibitor Control (Kojic Acid) 1 vial Orange K575-100-5
IV. User Supplied Reagents and Equipment:
96-well clear plate with flat bottom
Microplate reader
V. Storage and Handling:
Store kit at -20°C, protected from light. Avoid repeated freeze/thaw for all non-buffer components. Briefly centrifuge small vials prior to
opening. Read the entire protocol before performing the assay.
VI. Reagent Preparation & Storage
Tyrosinase Substrate: Dissolve the lyophilized tyrosinase substrate in 220 μl ddH2O. Use within two months. Keep on ice while in use.
Tyrosinase: Dissolve the lyophilized tyrosinase in 220 μl Tyrosinase Assay Buffer. Aliquot and store at -20°C. Avoid repeated freeze/thaw
cycles. Use within two months. Keep on ice while in use.
Tyrosinase Enhancer: Ready to use. Protect from light. Keep at room temperature while in use.
Inhibitor Control (Kojic Acid): Add 75 µl of ddH2O to make a stock solution of 10 mM Kojic Acid. Mix well. Make a 0.75 mM working
solution of Kojic Acid by adding 92.5 µl of ddH2O to 7.5 µl of 10 mM Kojic Acid Stock solution. Use within two months.
VII. Tyrosinase Inhibitor Screening Protocol:
1. Screening compounds, Inhibitor control and Blank Control Preparations: Dissolve test inhibitors into proper solvent. Dilute to 5X the
desired test concentration with Tyrosinase Assay Buffer before use. Add 20 μl diluted test inhibitors, Inhibitor Control working solution, or
Tyrosinase Assay Buffer into wells assigned as test inhibitors (Sample, S), Inhibitor Control (IC), or Tyrosinase Enzyme Control (EC) wells,
respectively. Additional wells with serial dilutions of the test inhibitors may be prepared at this time if desired, containing 20 µl in each
candidate well.
Note: Preferred final solvent concentration should not be more than 5% by volume. If solvent exceeds 5% include a Solvent Control to test
the effect of the solvent on enzyme activity.
2. Tyrosinase Enzyme Solution Preparation: For each well, prepare 50 μl Tyrosinase Enzyme Solution.
48 μl Tyrosinase Assay Buffer
2 μl Tyrosinase
Mix well & add 50 μl/well into wells containing test inhibitors, Inhibitor Control & Enzyme Control. Mix. Incubate for 10 min. at 25ºC.
3. Tyrosinase Substrate solution preparation: For each well, prepare 30 µl of Tyrosinase Substrate solution
23 μl Tyrosinase Assay Buffer
2 μl Tyrosinase Substrate
5 µl Tyrosinase Enhancer
Mix and add 30 μl of Tyrosinase Substrate Solution into each well. Mix well.
4. Measurement: Measure the absorbance in kinetic mode for 30-60 min. at 510 nm. Choose two time points (T1 & T2) in the linear range of
the plot and obtain the corresponding values for the Absorbance (Abs1 and Abs2).
5. Calculations: Calculate the slope for all samples, including Enzyme Activity Control (EC), by dividing the net ΔAbs (Abs2- Abs1) values by
the time ΔT (T2-T1). Calculate % Relative Inhibition as follows:
155 S. Milpitas Blvd., Milpitas, CA 95035 USA | T: (408)493-1800 F: (408)493-1801 | [Link] | tech@[Link]
FOR RESEARCH USE ONLY!
100
Residual Activity (%)
80
60
40
20
0
1 10 100
Kojic Acid (µM)
Figure: Inhibition of Tyrosinase Enzymatic Activity with Kojic Acid. Assay was performed following kit protocol.
VIII. RELATED PRODUCTS:
Asparaginase Activity Colorimetric/Fluorometric Assay Kit (K754) Glutamate Dehydrogenase Activity Colorimetric Assay Kit (K729)
Aspartate Aminotransferase Activity Colorimetric Assay Kit (K753) Arginase Activity Colorimetric Assay Kit (K755)
FOR RESEARCH USE ONLY! Not to be used on humans.
155 S. Milpitas Blvd., Milpitas, CA 95035 USA | T: (408)493-1800 F: (408)493-1801 | [Link] | tech@[Link]
