Blue Native PAGE Analysis of Bacterial Secretion Complexes
Bacterial protein secretion systems serve to translocate substrate proteins across up to three biological membranes, a task accomplished by hydrophobic, membrane-spanning macromolecular complexes. The overexpression, purification, and biochemical characterization of these complexes is often difficult, impeding progress in understanding the structure and function of these systems. Blue native (BN) polyacrylamide gel electrophoresis (PAGE) allows for the investigation of these transmembrane complexes right from their originating membranes, without the need for long preparative steps, and is amenable to the parallel characterization of a number of samples under near-native conditions. Here we present protocols for sample preparation, one-dimensional BN PAGE and two-dimensional BN/sodium dodecyl sulfate (SDS)-PAGE, as well as for downstream analysis by staining, immunoblotting, and mass spectrometry on the example of the type III secretion system encoded on Salmonella pathogenicity island 1.
Key wordsBacterial secretion systems Membrane proteins Blue native polyacrylamide gel electrophoresis Two-dimensional polyacrylamide gel electrophoresis Sucrose gradients Bacterial cell fractionation Type III secretion systems Salmonella typhimurium Escherichia coli
Work performed in the laboratory of SW was supported by the Alexander von Humboldt Foundation in the framework of the Sofja Kovalevskaja Award endowed by the Federal Ministry of Education and Research (BMBF) and in the framework of the Georg Forster Research Fellowships (to J.V.M.F.), and by the Deutsche Forschungsgemeinschaft (DFG) as part of the Collaborative Research Center (SFB) 766 Bacterial cell envelope, project B14.
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