Comparative analysis of cytoplasmic membrane proteomes of Escherichia coli using 2D blue native/SDS-PAGE
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Comparative analysis of cytoplasmic membrane proteomes of Escherichia coli using 2D blue native/SDS-PAGE. / Schlegel, Susan; Klepsch, Mirjam; Wickström, David; Wagner, Samuel; de Gier, Jan-Willem.
in: Methods Mol Biol, Jahrgang 619, 2010, S. 257-69.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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T1 - Comparative analysis of cytoplasmic membrane proteomes of Escherichia coli using 2D blue native/SDS-PAGE
AU - Schlegel, Susan
AU - Klepsch, Mirjam
AU - Wickström, David
AU - Wagner, Samuel
AU - de Gier, Jan-Willem
PY - 2010
Y1 - 2010
N2 - Two-dimensional blue native (2D BN)/SDS-PAGE is the method of choice for the global analysis of the subunits of complexes in membrane proteomes. In the 1st dimension complexes are separated by BN-PAGE, and in the 2nd dimension their subunits are resolved by SDS-PAGE. The currently available protocols result in the distortion of the 1st dimension BN-gel lanes during their transfer to the 2nd dimension separation gels. This leads to low reproducibility and high variation of 2D BN/SDS-gels, making 2D BN/SDS-PAGE unsuitable for comparative analysis. Here, we present a 2D BN/SDS-PAGE protocol where the 1st dimension BN-gel is cast on a GelBond PAG film. Immobilization prevents distortion of BN-gel lanes when they are transferred to the 2nd dimension, which lowers variation and greatly improves reproducibility of 2D BN/SDS-gels. The use of 2D BN/SDS-PAGE with an immobilized first dimension is illustrated by the characterization of the cytoplasmic membrane proteome of Escherichia coli cells overexpressing cytochrome bo (3).
AB - Two-dimensional blue native (2D BN)/SDS-PAGE is the method of choice for the global analysis of the subunits of complexes in membrane proteomes. In the 1st dimension complexes are separated by BN-PAGE, and in the 2nd dimension their subunits are resolved by SDS-PAGE. The currently available protocols result in the distortion of the 1st dimension BN-gel lanes during their transfer to the 2nd dimension separation gels. This leads to low reproducibility and high variation of 2D BN/SDS-gels, making 2D BN/SDS-PAGE unsuitable for comparative analysis. Here, we present a 2D BN/SDS-PAGE protocol where the 1st dimension BN-gel is cast on a GelBond PAG film. Immobilization prevents distortion of BN-gel lanes when they are transferred to the 2nd dimension, which lowers variation and greatly improves reproducibility of 2D BN/SDS-gels. The use of 2D BN/SDS-PAGE with an immobilized first dimension is illustrated by the characterization of the cytoplasmic membrane proteome of Escherichia coli cells overexpressing cytochrome bo (3).
KW - Cell Membrane
KW - Electrophoresis, Gel, Two-Dimensional
KW - Electrophoresis, Polyacrylamide Gel
KW - Escherichia coli
KW - Proteome
KW - Journal Article
KW - Research Support, Non-U.S. Gov't
U2 - 10.1007/978-1-60327-412-8_15
DO - 10.1007/978-1-60327-412-8_15
M3 - SCORING: Journal article
C2 - 20419415
VL - 619
SP - 257
EP - 269
JO - Methods Mol Biol
JF - Methods Mol Biol
SN - 1064-3745
ER -