Optimisation of Ammonium Sulfate for Protein Purification from Kedah-Kelantan Cattle (Bos Indicus) Placenta

The optimisation of ammonium sulfate, ((NH4)2SO4) was applied to purify protein based on solubility from Kedah-Kelantan cattle (Bos Indicus) placenta. Double precipitation was carried out by using different concentration of (NH4)2SO4. First precipitation used 0% (initial concentration), 20%, 40%, 60% and 80% of (NH4)2SO4 as final concentration. Meanwhile, second precipitation was started with 20%-40%, 40%-60%, 40%-80% and 60%-80% of (NH4)2SO4. In first precipitation, the extracted supernatant was added with different concentration of (NH4)2SO4. An incubation was applied for 2 hours at 4°C. The sample were centrifuged for 1 hour at 15,930 rpm. The supernatant from first precipitation was used in second precipitation. The (NH4)2SO4 was added to achieve required concentration. The sample was incubated for overnight at 4°C. Centrifugation was applied for 1 hour at 15,930 rpm. Dialysis was carried out to remove the salts from the sample. The precipitates from second precipitation were dissolved in 1ml of 0.01 M Tris-HCl buffer (pH 7.5) and extensively dialyzed against same buffer. The samples were centrifuged for 1 hour at 12,000 rpm. The supernatant was undergoes Bradford protein
assay. In the first precipitation, the concentration of protein was highest (22.92 mg/ml) at 0%-60% of (NH4)2SO4 with 40% concentration of unknown protein sample. The concentration of protein was significantly highest (25.09 ml/mg) at 40%-60% of (NH4)2SO4 with 20% concentration of unknown protein sample in second precipitation. These data showed that the concentration of (NH4)2SO4 play important role in purification protein based on solubility.