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Purification of serum proteins by ammonium sulfate fractionation and column chromatography
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Ammonium sulfate were added to aliquots of FBS at 30%, 50%, 70% and 90% saturation and incubated on ice for 30 min with occasional agitation. The precipitates were collected by centrifugation at 12000xg, 4 °C for 10 min, and dialyzed against PC buffer (20 mM HEPES, 50 mM NaCl, pH7.4) and stored at -80 °C before use. The fraction of 70% ammonium sulfate-saturated components was further purified by fast protein liquid chromatography (FPLC) Mono Q and Superose 12™ (S12) column chromatographies.
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