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Identification of Lipid Binding Modulators Using the Protein-Lipid Overlay Assay

  • Tuo-Xian Tang
  • Wen Xiong
  • Carla V. Finkielstein
  • Daniel G. S. Capelluto
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1647)

Abstract

The protein-lipid overlay assay is an inexpensive, easy-to-implement, and high-throughput methodology that employs nitrocellulose membranes to immobilize lipids in order to rapid screen and identify protein-lipid interactions. In this chapter, we show how this methodology can identify potential modulators of protein-lipid interactions by screening water-soluble lipid competitors or even the introduction of pH changes during the binding assay to identify pH-dependent lipid binding events.

Key words

Lipid-protein overlay assay Phospholipids Phosphoinositides Inositol 1,3-bisphosphate EEA1 FYVE Dishevelled-2 DEP Phafin2 Tollip pH effect 

Notes

Acknowledgments

We thank Janet Webster for critical reading and comments on the manuscript. We also thank Tiffany Radle and Morgan Vaughn for the optimization of the purification conditions of the GST-EEA1 FYVE domain. Work in the Capelluto laboratory is supported by the National Institutes of Health (NIAID).

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Tuo-Xian Tang
    • 1
    • 2
  • Wen Xiong
    • 1
    • 2
  • Carla V. Finkielstein
    • 3
  • Daniel G. S. Capelluto
    • 1
    • 2
  1. 1.Protein Signaling Domains Laboratory, Department of Biological Sciences, Biocomplexity InstituteVirginia TechBlacksburgUSA
  2. 2.Center for Soft Matter and Biological PhysicsVirginia TechBlacksburgUSA
  3. 3.Integrated Cellular Responses Laboratory, Department of Biological Sciences, Biocomplexity InstituteVirginia TechBlacksburgUSA

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