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Membrane Insertion of Small Proteins

  • Andreas Kuhn
  • Natalie Stiegler
  • Anne-Kathrin Schubert
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 619)

Abstract

Proteins that are less than 10 kDa in size are easily purified under denaturing conditions and can often be refolded by removal of the denaturing agents. The purified small membrane proteins are competent for membrane insertion when the denaturing agent is diluted out and a membranous system like liposomes or proteoliposomes is added. This system allows the characterization of the membrane insertion process at the molecular level. The insertion of the protein into proteoliposomes can be followed by protease digestion and Western blot analysis. Only if the antigenic region of the protein has translocated into the lumen of the proteoliposome it is protected from the protease. When combining this approach with fluorophores that are placed within the membrane protein, membrane insertion can also be followed by fluorescence correlation spectroscopy.

Key words

Pf3 coat protein M13 procoat protein YidC membrane insertase liposomes fluorescence labelling 

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Andreas Kuhn
    • 1
  • Natalie Stiegler
    • 1
  • Anne-Kathrin Schubert
    • 1
  1. 1.Institute of Microbiology and Molecular BiologyUniversity of HohenheimStuttgartGermany

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