Metodi elettroforetici bidimensionali applicati allo studio di diversi sistemi membranali
Author(s)
D'Amici, Gian Maria
Date Issued
April 4, 2008
Type
Doctoral Thesis
Abstract
Developments in methods and technologies have resulted in an expansion of the
purpose of biological studies from the reductionist biochemical analysis of single
proteins to proteome-wide measurements. Proteomics and other complementary
analysis methods are essential components of the emerging 'systems biology'.
Proteome analysis implies the ability to separate proteins as a first step prior the
characterization. Thus, the overall performance of the analysis strongly depends on
the performance of the separation tool, usually two dimensional electrophoresis.
This thesis gives an introduction to the proteomic technique and shows how twodimensional
electrophoresis works with membrane proteins. Different subjects and
biological systems were investigated using different separative two-dimensional
electrophoresis techniques. In chapter one, two-dimensional gel electrophoresis (2D
IEF-SDS-PAGE) and mass spectrometry were used to identify protein profile
changes in red blood cell membranes stored over time under atmospheric oxygen, in
the presence or absence of protease inhibitors. In chapter two, a new 3D native
electrophoretic protocol is proposed for an exhaustive separation and identification
of membrane proteins. This method is based on native liquid phase
isoelectrofocusing (N-LP-IEF) of protein complexes in the first dimension, followed
by blue native polyacrylamide gel electrophoresis (BN-PAGE) in the second
dimension, where both the pI and the molecular masses of protein complexes (2D
N-LP-IEF-BN) were used to separate them in their native form.
Additional information
Dottorato di ricerca in Genetica e biologia cellulare
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