2.6 Extraction of crude protein from bovine liver
Crude preparation of FAS enzyme was made according to modified protocol of Dulter et al . (1971). Liver from freshly slaughtered Indian water buffalo was collected and transported in ice bucket and kept at 4 °C. The liver was processed at the earliest by removing connective tissues and sliced into small pieces. About 750 g of liver was minced in a blender into suspension with 0.05 M Potassium phosphate buffer pH 7.4 containing 1 mM EDTA, to make up to a volume of 2.5 L. The thick solution was centrifuged at 7500 x g for 20 min to remove meat debris and supernatant was further centrifuged at 30000 x g for 30 min to get a translucent solution. The supernatant was further diluted to specific volume with 0.05 M Potassium phosphate buffer pH 7.4 with 1 mM EDTA and brought initially to 10% and then to 40% saturation with solid ammonium sulfate at 4 °C. The 40% ammonium sulfate fraction was centrifuged and protein precipitate was redissolved in 0.05 mM Tris.HCl buffer, pH 7.4, dialyzed against same buffer and used for further purification. Enzyme was further purified by ion exchange chromatography with DEAE cellulose column (Dulter et al ., 1971).