title: Two-dimensional maps in soft immobilized pH gradient gels: a new approach to the proteome of the Third Millennium.
abstract: Same major improvements in proteome analysis of cytosolic and membrane proteins by two-dimensional mapping are here reported. A much improved transfer of proteins from the first to the second dimensional sodium dodecyl sulfate (SDS)-gel is obtained by simply diluting the gel matrix, normally composed of 4%T polyacrylamide in all commercially available Immobiline strips down to as low as 3%T. In the analysis of total lysates of platelets, this augmented transfer has been evaluated as being 2-3 times higher than in standard 4%T gels. A second major improvement, in the case of analysis of membrane protein preparations, has been demonstrated to consist in a delipidation step in a tertiary solvent mixture composed of tri-n-butyl phosphate:acetone:methanol in a 1:12:1 ratio. By adopting this protocol, large amounts of spectrins (240-220 kDa, filamentous proteins of the red blood cell membranes) could be transferred vs. essentially none when delipidation was omitted. The present report also confirms the importance of a reduction and alkylation step of the protein sample prior to all electrophoretic steps, including focusing in the Immobiline gel, as recently reported by Herbert et al.
authors.affiliation: Nephrology Section, G. Gaslini Children's Hospital, Genova, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Candiano
authors.forename: Luca
authors.initials: L
authors.lastname: Musante
authors.forename: Maurizio
authors.initials: M
authors.lastname: Bruschi
authors.forename: Gian Marco
authors.initials: GM
authors.lastname: Ghiggeri
authors.forename: Ben
authors.initials: B
authors.lastname: Herbert
authors.forename: Francesca
authors.initials: F
authors.lastname: Antonucci
authors.forename: Pier Giorgio
authors.initials: PG
authors.lastname: Righetti
mesh_terms: D000180:Acrylic Resins; D001792:Blood Platelets / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004910:Erythrocyte Membrane / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000032:analysis*; D010449:Peptide Mapping / Q000379:methods; D020543:Proteome / Q000032:analysis*
chemical_list: D000180:Acrylic Resins; D008565:Membrane Proteins; D020543:Proteome; C016680:polyacrylamide gels