2.5 Preparation of bacterial FAS for FAS inhibition assay
Crude FAS containing protein fraction was isolated according to the modified protocol of Ahmad et al. (1981). Stock culture ofBacillus subtilis was inoculated in 100 mL LB broth pH 7.2 and cultured for 24 hrs at room temperature. It was used for further inoculation of four 1000 mL flasks with same media and culture conditions. The flasks were placed in a rotator shaker to ensure adequate suspension of cells. Bacteria were harvested during the late-exponential phase of growth by centrifugation at 10000 x g for 10 min and washed twice with 50 mM sodium phosphate buffer (pH 7.0). Cell pastes were stored at -20 °C until use.