To assay 17 b-HSD activity in lysates, cells were harvested 48h after transfection using PBS Enzyme Free Cell Dissociation Solution ( specialty Media Inc., Lavellette, NJ), frozen on dry ice and stored at -80 C.
Cell pellets were thawed on ice, resuspended in :
A typical assay for establish the apparent Vmax and Km of the Substrate ANDROST-4-ENE-3,17-DIONE of the normal & mutant HSD17B3 enzyme used: * of total protein in * Steroid substrate was added in different concent. , and NADPH was added to a final cocent. of 2mM. * Reactions were initiated by the addition of enzyme and were carried out for 25? 30?min at 37 ?C . A typical assay to establish the apparent pH of the normal & mutant HSD17B3 enzyme used: * Serial variation of pH (range4.5pH -8.5pH), for the reaction Buffer : . * Steroid substrate was added at final concent. of 5 mM ( where 1 mM were 14C- ANDROST-4-ENE-3,17-DIONE and 4mM were cold substrate). Transfection of DNA into 293T cells by using LIPOFECTAMINE PLUS Reagent package (LIFE TECHNOLOGIES Cat.No.10964-013): * The day before transfection, split and plating the cells, so the that they are 50%- 60% confluent the day of transfection. Avoid antibiotics at the time of transfection and during. * Culture vessel: * plasmid DNA ; * plasmid b-gal ; * PLUS reagent; * LIPOFECTAMINE PLUS Reagent; * Incubate at 37癈 at 5% CO2 for 3h * After 3h inc., increase volume of medium to normal volume * The cell extract were assayed for reporter gene activity 48h after the start of transfection.
Protocol