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.
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
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).
Bonham KS, Orzalli MH, Hayashi K et al (2014) A promiscuous lipid-binding protein diversifies the subcellular sites of toll-like receptor signal transduction. Cell 156:705–716CrossRefPubMedPubMedCentralGoogle Scholar
Nakamura Y, Andres F, Kanehara K et al (2014) Arabidopsis florigen FT binds to diurnally oscillating phospholipids that accelerate flowering. Nat Commun 5:3553PubMedPubMedCentralGoogle Scholar
Lee SA, Eyeson R, Cheever ML et al (2005) Targeting of the FYVE domain to endosomal membranes is regulated by a histidine switch. Proc Natl Acad Sci U S A 102:13052–13057CrossRefPubMedPubMedCentralGoogle Scholar
Xiao S, Brannon MK, Zhao X et al (2015) Tom1 modulates binding of Tollip to phosphatidylinositol 3-phosphate via a coupled folding and binding mechanism. Structure 23:1910–1920CrossRefPubMedGoogle Scholar