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.
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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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Pérez-Sancho, J., Schapire, A.L., Botella, M.A., Rosado, A. (2016). Analysis of Protein–Lipid Interactions Using Purified C2 Domains. In: Botella, J., Botella, M. (eds) Plant Signal Transduction. Methods in Molecular Biology, vol 1363. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3115-6_14
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DOI: https://doi.org/10.1007/978-1-4939-3115-6_14
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3114-9
Online ISBN: 978-1-4939-3115-6
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