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Blue Native PAGE Analysis of Bacterial Secretion Complexes.

Susann Zilkenat1, Tobias Dietsche1, Julia V Monjarás Feria1

  • 1Section of Cellular and Molecular Microbiology, Interfaculty Institute of Microbiology and Infection Medicine (IMIT), University of Tübingen, Elfriede-Aulhorn-Str. 6, 72076, Tübingen, Germany.

Methods in Molecular Biology (Clifton, N.J.)
|July 2, 2017
PubMed
Summary

Investigating bacterial protein secretion systems is challenging. Blue native polyacrylamide gel electrophoresis (BN-PAGE) offers a method to study these membrane complexes directly from bacterial membranes under native conditions.

Keywords:
Bacterial cell fractionationBacterial secretion systemsBlue native polyacrylamide gel electrophoresisEscherichia coliMembrane proteinsSalmonella typhimuriumSucrose gradientsTwo-dimensional polyacrylamide gel electrophoresisType III secretion systems

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Area of Science:

  • Microbiology
  • Biochemistry
  • Molecular Biology

Background:

  • Bacterial protein secretion systems are essential for translocating proteins across membranes.
  • Studying these complex, membrane-spanning structures is hindered by difficulties in overexpression and purification.
  • Understanding secretion system structure and function is crucial for bacterial pathogenesis research.

Purpose of the Study:

  • To present optimized protocols for analyzing bacterial transmembrane protein complexes.
  • To demonstrate the utility of Blue native polyacrylamide gel electrophoresis (BN-PAGE) for studying these systems.
  • To provide a framework for characterizing the type III secretion system from Salmonella.

Main Methods:

  • Sample preparation for membrane protein complex analysis.
  • One-dimensional BN-PAGE and two-dimensional BN/SDS-PAGE.
  • Downstream analyses including staining, immunoblotting, and mass spectrometry.

Main Results:

  • Established protocols for analyzing membrane protein complexes directly from bacterial membranes.
  • Demonstrated the effectiveness of BN-PAGE for near-native characterization of secretion systems.
  • Successfully applied the methods to the Salmonella type III secretion system.

Conclusions:

  • BN-PAGE is a powerful technique for studying bacterial protein secretion systems.
  • The presented protocols facilitate biochemical characterization of complex transmembrane machinery.
  • This approach aids in understanding the structure and function of essential bacterial virulence factors.