Non-classical 2-D electrophoresis.
Methods Mol Biol
; 564: 33-57, 2009.
Article
em En
| MEDLINE
| ID: mdl-19544016
ABSTRACT
Classical 2-D electrophoresis (IEF/SDS 2-DE) using isoelectric focusing (IEF) and SDS-PAGE for the second dimension offers very high resolution for the separation of complex protein mixtures, but hydrophobic proteins can aggregate and are considerably under-represented in these 2-D gels. Non-classical 2-DE, as described here, summarizes several heterogeneous techniques, some of which, like BAC/SDS 2-DE and doubled SDS-polyacrylamide gel electrophoresis (dSDS-PAGE), intend to isolate the difficult hydrophobic proteins that are not accessible by classical 2-DE. Other types of non-classical 2-DE start with 1-D separation of native proteins and complexes, like blue-native electrophoresis (BNE), clear-native electrophoresis (CNE), and high-resolution clear-native electrophoresis (hrCNE). These electrophoretic techniques can substitute for chromatographic isolation of protein complexes, and can even isolate supramolecular physiological assemblies. Subsequent resolution in second dimension can be denaturing to resolve the subunits of complexes, as exemplified with BNE/SDS 2-DE, or native like in BNE/BNE 2-DE (the latter using different cathode buffers for 1-D BNE and 2-D BNE). After isolation of highly pure membrane protein complexes by two native electrophoretic separations, the separation protocol may be finished by denaturing 2-DE like BAC/SDS or doubled SDS-PAGE. Thus, a four-dimensional electrophoretic system with minimal loss of protein results that is useful as an efficient micro-scale protein separation protocol, e.g. for mass spectrometric analyses.
Texto completo:
1
Bases de dados:
MEDLINE
Assunto principal:
Fragmentos de Peptídeos
/
Eletroforese em Gel Bidimensional
/
Proteínas Mitocondriais
/
Proteômica
Limite:
Animals
Idioma:
En
Revista:
Methods Mol Biol
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2009
Tipo de documento:
Article
País de afiliação:
Estados Unidos