Comparative Analysis of Cytoplasmic Membrane Proteomes of Escherichia coli Using 2D Blue Native/SDS-PAGE
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.
Key wordsMembrane protein membrane isolation protein complex two-dimensional blue native SDS-PAGE comparative proteomics E. coli
This research was supported by grants from the Swedish Research Council and The Center for Biomembrane Research, which is supported by the Swedish Foundation for Strategic Research.
- 6.Schamel, W. W. A. (2008) Two-dimensional blue native polyacrylamide gel elektrophoresis. Curr Protoc Cell Biol, Chapter 6:Unit 6.10.
- 9.Wagner, S., Klepsch, M., Schlegel, S., Appel, A., Draheim, R., Tarry, M., Högbom, M., van Wijk, K. J., Slotboom, D. J., Persson, J. O., and de Gier, J. W. (2008) Tuning Escherichia coli for membrane protein overexpression. PNAS 105, 14371–14376.Google Scholar
- 10.Klepsch, M. M., Schlegel, S., Wickström, D., Friso, G., van Wijk, K. J., Persson, J.-O., de Gier, J.-W., and Wagner, S. (2008) Immobilization of the first dimension in 2D Blue Native/SDS-PAGE allows the relative quantification of membrane proteomes. Methods 46, 48–53.Google Scholar
- 12.Reisinger, V., and Eichacker, L. A. (2008) Solubilization of membrane protein complexes for blue native PAGE. J Proteomics 171, 277–283.Google Scholar