| Assay Method Information | |
| | Evaluation of Inhibitory Activities Against GCS |
| Description: | (1) MaterialsA549 cells (ATCC, CCL-185)NBD C6-ceramide (Thermo Fisher, N1154)UDP-glucose (Sigma, U4625)Potassium chloride (Sigma, P9333)UltraPure™ 0.5 M EDTA (Invitrogen, 15575-038)BCA protein assay kit (Thermo Fisher, 23227)Ibiglustat (Shanghai Systeam Biochem Co., ltd, Genz-682452)HEPES (sigma, H3375)Protease/phosphatase inhibitor cocktail (CST, 5872s)DMEM (GIBCO, 11995-065)FBS (GIBCO, 16000-044)Antibiotic-Antimycotic (100×) (GIBCO, 15240-112)200 mM L-glutamine (GIBCO, 25030081)PBS (GIBCO, 10010-023)0.25% Trypsin-EDTA (GIBCO, 25200-056)Dimethyl sulfoxide (Sigma, 34869)2-propanol, HPLC grade (Burdick & Jackson, AH323-4)Hexane, HPLC grade (Burdick & Jackson, AH216-4)Chloroform (Sigma, C2432)Methanol (Merck, 1.06009.1011)(2) Protocol<1> Preparation of Cell LysatesA549 cells (ATCC, CCL-185) were cultured in a DMEM medium supplemented with 10% fetal bovine serum (FBS), 1× antibiotic-antimycotic, and 1×L-glutamine, in an incubator at 37° C. and 5% CO2. After the cells attached to the culture dish were washed with phosphate buffered saline (PBS), the cells were scraped off with a cell scraper and then centrifuged (4000 rpm, 3 min, 4° C.) to collect the cells in a 50 ml tube. The cell pellets were suspended in a lysis buffer (50 mM HEPES, pH 7.3, containing 1× the protease/phosphatase inhibitor cocktail), lysed by sonication, and then the lysate was centrifuged (13000 rpm, 10 min, 4° C.). The obtained supernatant was used for the quantitative analysis of proteins. The amount of proteins was measured using the BCA protein assay kit, using bovine serum albumin as a standard. |
| Affinity data for this assay | |
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