Separation of Proteins by Aqueous Two-phase Extraction System Combined with Liquid Chromatography

A method was developed for the enrichment and separation of proteins by an aqueous two-phase extraction system (ATPS) of PEG/(NH4)2SO4 combined with high-performance liquid chromatography (HPLC). The effects of the type and the concentration of inorganic salts, PEG molecular weight, pH value, temperature and NaCl on the formation of two phases and partition of cytochrome c, lysozyme, trypsin, bovine serum albumin and myoglobin were investigated, respectively. The results showed that five proteins were enriched mainly in bottom phase in 15% PEG-4000/10% (NH4)2SO4 at room temperature and pH from 3.5 to 9.0. High-abundance proteins in serum were also distributed in two phases and the bottom phase had larger amount under the same conditions, which could be extracted and separated by optimized ATPS. The proteins were collected on the basis of their retention time in HPLC, and could be removed from serum samples.