title: Solubilization methods and reference 2-DE map of cow milk fat globules.
abstract: Milk fat globules (MFGs) are secretory vesicles assembled and secreted by mammary epithelial cells during lactation. They consist of fat globules surrounded by a lipid bilayer membrane which is derived from the apical membrane of the lactating cells. MFGs contain, besides lipids, proteins from the apical plasma membrane and from the cytoplasmatic material. Their peculiar vesicle nature makes them a suitable and easily available source of biological material in monitoring the physiopathological state of the mammary gland. Unfortunately, the conspicuous lipidic component of MFGs consistently limits protein extraction and purification for MFG proteomic investigations. This work deals with the development of a suitable procedure for protein extraction from the cow MFGs in order to qualitatively and quantitatively improve 2-D electropherograms of the MFG. MFGs were purified from raw milk by centrifugation and then delipidated/precipitated. The resulting protein pellets were solubilised using four different 2-D SDS PAGE compatible lysis buffers. Applied methodological procedures for protein extraction and evaluation of the resulting 2-D protein-pattern are presented and discussed. Using these procedures a reference 2-D map of cow milk fat globules is also reported. The majority of the obtained identifications was represented by proteins involved in lipid synthesis or in fat globule secretion.
authors.affiliation: Functional Proteomics Laboratory, Department of Molecular Biology, Siena University, Siena, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Bianchi
authors.forename: Michele
authors.initials: M
authors.lastname: Puglia
authors.forename: Claudia
authors.initials: C
authors.lastname: Landi
authors.forename: Silvia
authors.initials: S
authors.lastname: Matteoni
authors.forename: Daniele
authors.initials: D
authors.lastname: Perini
authors.forename: Alessandro
authors.initials: A
authors.lastname: Armini
authors.forename: Margherita
authors.initials: M
authors.lastname: Verani
authors.forename: Claudia
authors.initials: C
authors.lastname: Trombetta
authors.forename: Patrizia
authors.initials: P
authors.lastname: Soldani
authors.forename: Paola
authors.initials: P
authors.lastname: Roncada
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Greppi
authors.forename: Vitaliano
authors.initials: V
authors.lastname: Pallini
authors.forename: Luca
authors.initials: L
authors.lastname: Bini
mesh_terms: D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006017:Glycolipids / Q000737:chemistry*; D006023:Glycoproteins / Q000737:chemistry*; D066292:Lipid Droplets; D008055:Lipids / Q000737:chemistry; D008892:Milk / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D016622:Silver Staining / Q000379:methods
chemical_list: D003902:Detergents; D006017:Glycolipids; D006023:Glycoproteins; D008055:Lipids; D011506:Proteins; D020543:Proteome; C110367:milk fat globule