title: Straightforward isolation of phosphatidyl-ethanolamine-binding protein-1 (PEBP-1) and ubiquitin from bovine testis by hydrophobic-interaction chromatography (HIC).
abstract: Isolation of phosphatidyl-ethanolamine-binding protein-1 (PEBP-1) from bovine brain was described almost three decades ago but it required a large number of steps to reach high purity. After the fractionation of bovine testis proteins by ammonium sulfate precipitation we found that PEBP-1, detected by Western blotting, was among the very few proteins still soluble at 80% ammonium sulfate saturation (3.2M). This soluble fraction (S80) was directly loaded onto a phenyl sepharose column equilibrated at the same ammonium sulfate concentration (3.2M). A stepwise elution of the retained material at 1.0, 0.5, 0.2, 0.1M ammonium sulfate in ammonium hydrogen carbonate was performed and then with ammonium hydrogen carbonate alone and finally with 50% ethylene glycol. All fractions were analyzed by SDS-PAGE and Western blotting and the fractions containing PEBP-1 was further fractionated by size exclusion chromatography on a HR75 Superdex column permitting the isolation of ubiquitin in addition to PEBP-1 as demonstrated by Western blotting and mass spectrometry. This study shows the feasibility of hydrophobic interaction chromatography (HIC) on phenyl sepharose at a very high ammonium sulfate concentration (3.2M; 80% saturation) to efficiently purify the proteins that are still soluble in these extreme conditions.
authors.affiliation: Institut National de Recherche Agronomique, Centre National de Recherche Scientifique (CNRS), Physiologie de Reproduction et des Comportements Unit, 37380 Nouzilly, France.
authors.forename: Maya
authors.initials: M
authors.lastname: Haj Hassan
authors.forename: Danièle
authors.initials: D
authors.lastname: Klett
authors.forename: Claire
authors.initials: C
authors.lastname: Cahoreau
authors.forename: Yves
authors.initials: Y
authors.lastname: Combarnous
mesh_terms: D000645:Ammonium Sulfate / Q000737:chemistry; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002850:Chromatography, Gel; D002853:Chromatography, Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D057927:Hydrophobic and Hydrophilic Interactions; D008297:Male; D049488:Phosphatidylethanolamine Binding Protein / Q000737:chemistry / Q000302:isolation & purification*; D013737:Testis / Q000737:chemistry*; D025801:Ubiquitin / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D049488:Phosphatidylethanolamine Binding Protein; D025801:Ubiquitin; D000645:Ammonium Sulfate