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Analysis of Protein–Lipid Interactions Using Purified C2 Domains

  • Jessica Pérez-Sancho
  • Arnaldo L. Schapire
  • Miguel A. Botella
  • Abel RosadoEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1363)

Abstract

C2 domains (C2s) are regulatory protein modules identified in eukaryotic proteins targeted to cell membranes. C2s were initially characterized as independently folded Ca2+-dependent phospholipids binding domains; however, later studies have shown that C2s have evolutionarily diverged into Ca2+-dependent and Ca2+-independent forms. These forms interact and regulate their affinity to diverse lipid species using different binding mechanisms. In this protocol we describe a biochemical approach to produce, purify, and solubilize functional C2 domains bound to GST for the identification of their putative Ca2+-dependent and Ca2+-independent lipid-binding partners.

Key words

C2 domain Ca2+-dependent lipid binding Solubility Protein-lipid overlay assay Multilamellar vesicles 

Notes

Acknowledgments

This work was supported by the FPI-BES 2012-052324 from Spanish MICINN (to JPS), BIO2011-23859 cofinanced by the European Regional Development Fund and by Grant no. P07-CVI-03021 by La Consejería de Innovación Ciencia y Empresa-La Junta de Andalucía confinanced by the European Regional Development Fund (to MAB), and the NSERC Discovery Grant RGPIN-2014-06468 and NSERC Canada Research Chairs program (to AR).

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Jessica Pérez-Sancho
    • 1
  • Arnaldo L. Schapire
    • 2
  • Miguel A. Botella
    • 1
  • Abel Rosado
    • 3
    Email author
  1. 1.Departamento de Biología Molecular y BioquímicaInstituto de Hortofruticultura Subtropical y Mediterránea (IHSM-UMA-CSIC)MálagaSpain
  2. 2.Center for Research in Agricultural Genomics CRAG (CSIC-IRTA-UAB-UB)BarcelonaSpain
  3. 3.Department of Botany, Faculty of SciencesUniversity of British ColumbiaVancouverCanada

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