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Assay Method Information

Assay Name:  Inhibitory Activity Assay
Description:  Expression vector each expressing a gene was inserted using Lipofectamine 2000 (Invitrogen) to cultured cell line HEK293 cell derived from human fetus renal cell. The resistant strains were selected using G418, and among them, the strains showing human LAT-1- or LAT-2-specific uptake of amino acid were established as stably expressing cell lines, respectively.The above-mentioned stably expressing cells were plated on a 24-well collagen plate at 1.2105 cells/well, and, after 48 hr, the cells were washed (3) with uptake buffer (Na2+-free Hank's balanced salt solution (HBSS) pH7.4) kept at 37 C. Each test compound (0.1, 1, 3, 10, 30, 100, 300 and 1000 μM) was added thereto, and the mixture was kept at 37 C. for 3 min. [14C]L-leucine or alanine (1 μM) was added thereto, and [14C]L-leucine or alanine (1 μM) was uptaken for 1 min. The mixture was washed with ice-cold uptake buffer (3). Then, the cells were dissolved in 0.1M NaOH aqueous solution (500 μL), and the protein concentration was measured using the 20 μL of the solution.
Affinity data for this assay
 

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Last update November 1, 2007
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