Tyrosinase

Manufactured by Abcam

Sourced in United States, United Kingdom

Tyrosinase is an enzyme that catalyzes the oxidation of tyrosine to dopaquinone, a key step in the melanin biosynthesis pathway. It is a copper-containing monooxygenase enzyme that is found in various organisms, including plants, fungi, and animals.

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Lab products found in correlation

Pvdf membrane, by Merck Group (2 mentions) α tubulin, by Gentex (2 mentions) Tyrp1, by Abcam (2 mentions) Hrp polymers, by Biocare Medical (2 mentions) Pigf1r, by Abcam (2 mentions) Pgam1, by Proteintech (2 mentions) P akt, by Cell Signaling Technology (2 mentions) Dab substrate, by Thermo Fisher Scientific (2 mentions) Trp 1, by Abcam (2 mentions) Gapdh sc 32233, by Santa Cruz Biotechnology (1 mentions) β catenin 71 2700, by Thermo Fisher Scientific (1 mentions) Ripa buffer, by Merck Group (1 mentions) 2 d quant kit, by GE Healthcare (1 mentions) Odyssey infrared imaging system, by LI COR (1 mentions) Total erk, by Cell Signaling Technology (1 mentions) Proliferating cell nuclear antigen (pcna), by Abcam (1 mentions) Phospho erk, by Cell Signaling Technology (1 mentions) Sox10, by Abcam (1 mentions) P creb, by Cell Signaling Technology (1 mentions) Hsp90, by Santa Cruz Biotechnology (1 mentions)

Spelling variants of this product

Tyrosinase Activity Assay Kit (7 citations) Tyrosinase Inhibitor Screening Kit (4 citations) Rabbit anti-tyrosinase (4 citations)

11 protocols using tyrosinase

Melanogenesis Regulation by TCQA and α-MSH

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HEM and B16F10 were seeded at density of 5 × 10⁵ cells per 100 mm petri dish and then treated with 0, 10, and 25 μM of TCQA, and 200 nM of α-MSH. After 4, 8, 12, 24, and 48 h, total protein extraction was achieved using radioimmunoprecipitation assay (RIPA) buffer (Sigma, St. Louis, United States) and protease inhibitor following the manufacturer’s instructions.
The quantification was assessed using a 2-D Quant kit according to manufacturer’s instructions (GE Healthcare, Chicago, United States).
The protein sample (15 μg/well) was separated in 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, NJ, United States). After being blocked, the membranes were incubated with primary antibodies against β-catenin 71–2700 (Thermo Fisher Scientific, MA, United States), Mitf (Abcam, Rockford, United States), tyrosinase (Abcam, Rockford, United States), and Tyrp1 sc-166857, Dct sc-74439, and GAPDH sc32233 (Santa Cruz Biotechnology, TX, United States). After overnight incubation at 4°C, the second antibody goat anti-rabbit IRDye 800 CW or IRDye 680 LT goat anti-mouse was added. Then the expression was detected using LI-COR Odyssey Infrared Imaging System (LI-COR, NE, United States).

Bejaoui M., Villareal M.O, & Isoda H. (2020). 3,4,5-Tri-O-Caffeoylquinic Acid Promoted Hair Pigmentation Through β-Catenin and Its Target Genes. Frontiers in Cell and Developmental Biology, 8, 175.

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Melanocyte Protein Expression Analysis

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In this experiment, the following antibodies were used: tyrosinase, Tyrp1, DCT, MITF, SOX10, Pmel, Ki67, and PCNA were obtained from Abcam (Cambridge, UK). Total ERK, phospho-ERK, total CREB, and pCREB were purchased from Cell Signaling Technology (Danvers, MA, USA). Another antibody for tyrosinase (Santa Cruz, Dallas, TX, USA) was also used to confirm bands. Antibody for MLANA was obtained from Cell Marque (Rocklin, CA, USA). α-tubulin (Gentex, Holland, MI, USA) and HSP90 (Santa Cruz) were used as internal loading controls. Secondary antibodies used for western blotting were as follows: goat anti-rabbit IgG- horseradish peroxidase (HRP) (1:5,000), goat anti-mouse IgG-HRP (1:5,000), and mouse anti-goat IgG-HRP (1:5,000).

Yoo H., Lee H.R., Kim K.H., Kim M.A., Bang S., Kang Y.H., Kim W.H., Song Y, & Chang S.E. (2021). CRTC3, a sensor and key regulator for melanogenesis, as a tunable therapeutic target for pigmentary disorders. Theranostics, 11(20), 9918-9936.

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Immunohistochemical Analysis of Tumor Markers

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Tumors were fixed in formalin for 24 h, paraffin embedded, sectioned, and stained according to standard procedures. Briefly, endogenous peroxidases were inactivated by 3% hydrogen 673 peroxide. Non-specific signals were blocked using 3% BSA, 10% goat serum in 0.1% Triton X-100. After antigen retrieval in citrate buffer, slides were stained using respective antibodies overnight at 4°C, [ ENO1 (Proteintech, #11204–1-AP), PGK1 (Proteintech #17811–1-AP), PGAM1 (Proteintech, #16126–1-AP), Tyrosinase (Abcam, ab738), pAKT (Cell Signaling, #9271), pIGF1R (Abcam, ab39398)]. After overnight incubation, the slides were washed and incubated with secondary antibody (HRP-polymers, Biocare Medical) for 30 min at room temperature. The slides were washed three times and stained with DAB substrate (ThermoFisher Scientific). The slides were then counterstained with hematoxylin and mounted with mounting medium.

Maitituoheti M., Keung E.Z., Tang M., Yan L., Alam H., Han G., Singh A.K., Raman A.T., Terranova C., Sarkar S., Orouji E., Amin S.B., Sharma S., Williams M., Samant N.S., Dhamdhere M., Zheng N., Shah T., Shah A., Axelrad J.B., Anvar N.E., Lin Y.H., Jiang S., Chang E.Q., Ingram D.R., Wang W.L., Lazar A., Lee M.G., Muller F., Wang L., Ying H, & Rai K. (2020). Enhancer Reprogramming Confers Dependence on Glycolysis and IGF Signaling in KMT2D Mutant Melanoma. Cell reports, 33(3), 108293.

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Immunohistochemical Analysis of Melanocyte Markers

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Tissue samples were obtained from the mice, put in cryomolds with embedding medium and frozen at −80°C. Samples were sliced (7 μm thick) and then fixed with 4% paraformaldehyde and 0.1% Triton X-100 for 10 min. After 1 h of preparation with 5% normal donkey serum (Jackson ImmunoResearch), they were incubated at 4°C overnight with antibodies for tyrosinase (1:200 dilution; Abcam), MITF (1:200 dilution; Santacruz), MC1R (1:200 dilution; Abcam) and c-kit (1:200 dilution; Abcam) and with secondary antibody (1:100; Molecular Probes) for 1 h. The slides were counterstained with DAPI for 10 min. Immunohistochemistry was conducted in duplicate. The expression level of protein was quantified using the Image-J program.

Kwack M.H., Lee S., Lee E.H., Ha G.U., Kim G.H, & Lee W.J. (2022). Effect of a Precision Cryotherapy Device with Temperature Adjustability on Pigmentation. Indian Journal of Dermatology, 67(2), 204.

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Hesperidin Modulation of Melanogenesis

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Hesperidin (Figure 6), α-MSH, MTT and L-dihydroxyphenylalanine (L-DOPA) were purchased from Sigma-Aldrich Chemicals Co. (St. Louis, MO, USA). Antibodies recognizing β-actin, phospho-Erk1/2 (p-Erk1/2) and phospho-Akt (p-Akt) were obtained from Cell Signaling Technology (Danvers, MA, USA). MITF, tyrosinase, TRP-1, and TRP-2 antibodies were purchased from Abcam Biotechnology (Abcam, Cambridge, UK). PD98059 (a specific inhibitor of Erk1/2) and LY294002 (a specific inhibitor of PI3K) were purchased from Sigma-Aldrich Chemicals Co. (St. Louis, MO, USA).

Lee H.J., Lee W.J., Chang S.E, & Lee G.Y. (2015). Hesperidin, A Popular Antioxidant Inhibits Melanogenesis via Erk1/2 Mediated MITF Degradation. International Journal of Molecular Sciences, 16(8), 18384-18395.

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Protein Extraction and Western Blotting

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Total protein was extracted with RIPA buffer (Beyo-time) and quantified by the BCA protein detection kit (Thermo Fisher) in accordance with the manufacturer’s instruction. Protein was electrophoresed on 10% SDS-PAGE and therewith transferred to PVDF membranes (Millipore). Afterwards, membranes were sealed with 5% nonfat milk for 1 h and treated with the specific primary antibodies for tyrosinase (1：1000, abcam, MA, USA), Mitf (1：200, abcam), FTO (1：1000, abcam) and GAPDH (1：2000, abcam) at 4℃ overnight. Following conjugation to HRP-labeled secondary antibody, the bands were visualized by ECL reagent (Thermo Fisher).

Shang Z., Feng H, & Xia L. (2023). The Suppression Effects of Fat Mass and Obesity Associated Gene on the Hair Follicle-Derived Neural Crest Stem Cells Differentiating into Melanocyte by N6-Methyladenosine Modifying Microphthalmia-Associated Transcription Factor. International Journal of Stem Cells, 16(2), 135-144.

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Immunohistochemical Analysis of Tumor Markers

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Oxidative Stress and Tyrosinase in UV-B Exposure

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NADPH oxidase (Abcam, Waltham, MA, USA) and superoxide dismutase (SOD; Abcam) activity in the UV-B-radiated HEKn cells and skin tissue of each group were determined by using appropriate kits, following the manufacturers’ instructions.
Tyrosinase activity (Abcam) in the UV-B- or compound-conditioned medium-treated HEMn cells and skin tissue of each group was determined by using appropriate kits, following the manufacturers’ instructions.

Kim H.M., Byun K.A., Oh S., Yang J.Y., Park H.J., Chung M.S., Son K.H, & Byun K. (2022). A Mixture of Topical Forms of Polydeoxyribonucleotide, Vitamin C, and Niacinamide Attenuated Skin Pigmentation and Increased Skin Elasticity by Modulating Nuclear Factor Erythroid 2-like 2. Molecules, 27(4), 1276.

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Protein Expression Analysis by Western Blot

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For the Western blotting, the cells were washed once with cold PBS and lysed in a protein lysis buffer (1% SDS in 10 mM Tris and 5 mM EDTA, pH 7.4) followed by incubation at 98 °C for 10 min. The extracted protein samples from the cells or human skin tissues were separated by 8% SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes (GE Healthcare Life Science, Amersham, Marlborough, MA, USA), blocked with Tris-buffered saline containing 0.1% Tween 20 and 5% BSA. The membranes were then subjected to immunoblotting with the antibodies listed below.
Tyrosinase, Tyrp1, and DCT antibodies were purchased from Abcam (Cambridge, UK). The MITF was from Cell Signaling Technology (Danvers, MA, USA). The antibodies against the total and phosphor form of AKT, ERK, β-catenin, CREB, GSK-β, p38 MAPK, SAPK/JNK, and mTOR were purchased form Cell Signaling Technology. Antibodies against HSP 90α/β (Santa Cruz, Dallas, TX, USA) or α-tubulin (Gentex, Holland, MI) were used as an internal loading control.

Lee J.H., Choi M.E., An H., Moon J.W., Yeo H.J., Song Y, & Chang S.E. (2022). BCI-215, a Dual-Specificity Phosphatase Inhibitor, Reduces UVB-Induced Pigmentation in Human Skin by Activating Mitogen-Activated Protein Kinase Pathways. Molecules, 27(17), 5449.

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Quantitative analysis of VEGF signaling proteins

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Cells were treated with VEGF165 and TA when grown to 80% confluent. Total cellular protein was extracted with lysis buffer (Beyotime Biotechnology, Beijing, China). An aliquot of proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto the polyvinylidene difluoride membranes (Millipore, Bedford, MA). After blocking for 30 min, the membrane was incubated with primary antibodies for 2 h at room temperature, and then conjugated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson Laboratories, West Grove, PA, USA) (1:5000 dilution) for 1 h. Immunoreactivity was detected by ECL plus luminal solution (Amersham Biosciences, Piscataway, NJ, USA). Immunoreactive bands were detected using an enhanced chemiluminescence system (Millipore). Protein density was measured by a Bio-Rad XRS chemiluminescence detection system (Bio-Rad, USA). The blots shown are representative of at least three repeats. The primary antibodies used in this study were antibodies against VEGFR-1, VEGFR2, NRP-1 (1:500-1:1000 dilution) (Santa Cruz, CA, USA), phospho-VEGFR-1 (Y1213), phospho-VEGFR-2 (Tyr1175) (1:500 dilution) (Cell Signaling Technology, Danvers, MA, USA), MITF, Tyrosinase, Trp-1, Trp-2 (1:1000 dilution) and β-actin (1:5000 dilution) (Abcam, Shanghai, China).

Zhu J.W., Ni Y.J., Tong X.Y., Guo X., Wu X.P, & Lu Z.F. (2020). Tranexamic Acid Inhibits Angiogenesis and Melanogenesis in Vitro by Targeting VEGF Receptors. International Journal of Medical Sciences, 17(7), 903-911.

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