Tyrosinase activity was determined with Tyrosinase Activity Assay Kit (Abcam, cat. no. ab252899), following the recommended protocols. Tyr-L B16 cells and Tyr-H B16 cells were cultured in DMEM medium (with addition of 10% fetal bovine serum, 1% penicillin & streptomycin and 1% glutamax) in a 5% CO2 humidified atmosphere at 37 °C. Parental B16 melanoma cells were cultured as above with or without 2-ETZ or L-tyrosine addition for another 24 h before determination of protein expression and tyrosinase activity.
Tyrosinase activity assay kit
Manufactured by Abcam
Sourced in United Kingdom
The Tyrosinase Activity Assay Kit is a colorimetric assay designed to measure the activity of the enzyme tyrosinase. Tyrosinase is a key enzyme involved in the production of melanin pigments. The kit provides a simple and convenient method to quantify tyrosinase activity in various biological samples.
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Lab products found in correlation
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Model 680 microplate reader, by Bio-Rad (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Glucose assay kit, by Abcam (1 mentions)
Copper assay kit, by Merck Group (1 mentions)
Lipid peroxidation mda assay kit, by Abcam (1 mentions)
7 protocols using tyrosinase activity assay kit
1
Tyrosinase Activity Assay in B16 Melanoma Cells
2
Measuring Melanogenesis and Signaling
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B16F10 cells and dorsal skin tissues were lysed, and tyrosinase activity and the levels of nitric oxide (NO) and cyclic AMP (cAMP) were then determined using the Tyrosinase Activity Assay Kit (Abcam, Cambridge, UK), Nitric Oxide Assay Kit (Abcam, Cambridge, UK), and cAMP ELISA Kit (Enzo Life Sciences, PA, USA), respectively, according to the manufacturer protocols.
3
Quantification of Tyrosinase Activity in Cultured Melanocytes
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The activity of tyrosinase in cultured melanocytes was measured using the Tyrosinase Activity Assay Kit (ab25899; Abcam, Tokyo, Japan) after the indicated treatments. Briefly, melanocytes (5 × 105 cells) were homogenized with 500 μL of ice-cold Tyrosinase Assay buffer to perform lysis and kept on ice for 10 min, and the mixture was centrifuged at 10,000× g for 15 min at 4 °C. The tyrosinase activity in the supernatant was determined spectrophotometrically with absorbance at 510 nm using a Model 680 Microplate Reader (Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer’s protocol.
4
Measuring Cellular Tyrosinase Activity
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The cellular tyrosinase activity were measured using tyrosinase Activity Assay Kit (Abcam, Cambridge, UK) and a slight modification of a previously reported method [40 (link)]. B16-F10 cells were seeded in 60 mm dishes (2 × 105 cells/dish) for 24 h and treated with CFS (0.5 and 1% (v/v)) or arbutin (200 µM) for 40 h in the presence or absence of 200 nM α-MSH, harvested by trypsinization, sonicated in assay buffer (Abcam, Cambridge, UK), and centrifuged at 12,000 rpm for 20 min. The protein concentration was determined by the Pierce™ BCA Protein Assay Kit (Thermo Fischer Scientific). The reaction mixture consisting of 20 µg protein and 80 µL of 2 mg/mL L-DOPA (in 0.1 M sodium phosphate buffer, pH 6.8) was added to each well of a 96-well plate. After incubation at 37 °C for 1 h, the optical density at 492 nm was measured using a microplate reader.
5
Tyrosinase Activity Quantification
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A tyrosinase activity assay (Tyrosinase activity assay kit; Abcam, Cambridge, UK) was performed according to the manufacturer’s instructions. Briefly, after dispensing the same amount of sample in a 96-well plate, 50 μL of the reaction mix included in the kit was added to the wells and thoroughly mixed, and the absorbance was measured at 510 nm in 30 s intervals during the reaction at 37 °C.
6
Measuring Cellular Tyrosinase Activity
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The enzymatic activity of cellular TYR was quantified using the Tyrosinase Activity Assay Kit (Abcam # ab252899; Cambridge, UK) according to the manufacturer's instructions.
7
Multiparametric Evaluation of Oxidative Stress
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Tyrosinase concentrations were assayed using (Tyrosinase Activity Assay Kit, Abcam, USA, Cat. No. ab252899) following colorimetric method Qu et al. [16] , Serum copper was determined using (Copper Assay Kit, Sigma-Aldrich , USA, Cat. No. MAK127) following the method of Changfeng et al. [17] . Serum fasting glucose levels were determined using (Glucose Assay Kit, Abcam, USA, Cat. No. ab65333 ) as described by Saw et al. [18] , Total Antioxidant (TAC) in liver tissue was determined using (Total Antioxidant Assay Kit, Elabscience Biochemistry Inc, USA, Cat.
No. E-BC-K136-S) following colorimetric assay of Marziyeh et al. [19] . Malondialdehyde (MDA) in liver tissue was assayed using (Lipid Peroxidation (MDA) Assay Kit, Abcam, USA, Cat. No. ab118970) using a method adapted to Wang et al. [20] . Hemoglobin, White Blood Cells, and Platelet counts were determined by the automatic cell counter as previously described [21] .
No. E-BC-K136-S) following colorimetric assay of Marziyeh et al. [19] . Malondialdehyde (MDA) in liver tissue was assayed using (Lipid Peroxidation (MDA) Assay Kit, Abcam, USA, Cat. No. ab118970) using a method adapted to Wang et al. [20] . Hemoglobin, White Blood Cells, and Platelet counts were determined by the automatic cell counter as previously described [21] .
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