| Assay Method Information | |
| | Inhibition Assay |
| Description: | The activity of the inhibitors is measured using a lysate of B16F1 cells (murine melanoma line). In the presence of the L-tyrosine substrate, the tyrosinase present in these cells catalyses the hydroxylation of L-tyrosine to give L-DOPA and then the oxidation of the L-DOPA to give dopaquinone. In the presence of MBTH (3-methyl-2-benzothiazolinone hydrazone), the dopaquinone is trapped so as to form a pink complex which absorbs at 520 nm.The B16F1 cells are cultured in DMEM medium+10% foetal calf serum+10-9 M alpha -MSH for 4 days at 37 C. under 7% CO2. They are treated with trypsin, washed in PBS, counted and pelleted. The pellet is taken up at 107 cells/ml in lysis buffer (10 mM sodium phosphate, pH 6.8-1% Igepal) and the suspension is treated with ultrasound for 10 seconds. After centrifugation for 30 minutes at 4000 rpm, the supernatant obtained constitutes the cell lysate used as tyrosinase source in the enzymatic assay. The assays are carried out in duplicate in 384-well plates. |
| Affinity data for this assay | |
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