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Analyzing Protein–Phosphoinositide Interactions with Liposome Flotation Assays

  • Ricarda A. Busse
  • Andreea Scacioc
  • Amanda M. Schalk
  • Roswitha Krick
  • Michael Thumm
  • Karin KühnelEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1376)

Abstract

Liposome flotation assays are a convenient tool to study protein–phosphoinositide interactions. Working with liposomes resembles physiological conditions more than protein–lipid overlay assays, which makes this method less prone to detect false positive interactions. However, liposome lipid composition must be well-considered in order to prevent nonspecific binding of the protein through electrostatic interactions with negatively charged lipids like phosphatidylserine. In this protocol we use the PROPPIN Hsv2 (homologous with swollen vacuole phenotype 2) as an example to demonstrate the influence of liposome lipid composition on binding and show how phosphoinositide binding specificities of a protein can be characterized with this method.

Key words

Analytical ultracentrifuge Small unilamellar vesicles Protein–lipid overlay assay PROPPIN Hsv2 

Notes

Acknowledgements

We thank Geert van den Bogaart for advice and discussions. This work was supported by a SFB860 grant to M.T. and K.K.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Ricarda A. Busse
    • 1
  • Andreea Scacioc
    • 1
  • Amanda M. Schalk
    • 1
    • 2
  • Roswitha Krick
    • 3
  • Michael Thumm
    • 3
  • Karin Kühnel
    • 1
    Email author
  1. 1.Department of NeurobiologyMax-Planck-Institute for Biophysical ChemistryGöttingenGermany
  2. 2.Department of Biochemistry and Molecular GeneticsUniversity of Illinois at ChicagoChicagoUSA
  3. 3.Institute of Cellular BiochemistryUniversity Medicine, Georg-August UniversityGöttingenGermany

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