Abstract
We adapted an efficient cell-free protein synthesis–based protocol for the production of lipid-binding proteins. The experimental procedures are based on the following steps: (1) cell-free synthesis of soluble, lipid-binding proteins fused to small tags; (2) analysis by dot blot of the accessibility of antibodies to the small tags. (3) protein lipid overlay assay with, immunodetection of bound protein by either chemiluminescence or fluorescence. We also provide a fast and inexpensive protocol for homemade lipid nitrocellulose strips spotted with acidic lipids (mostly phosphoinositides) extracted from plant tissues. These homemade lipid strips can be used for preliminary screen and characterization of putative phosphoinositide-binding proteins.
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This work was supported by NSF grant MCB1614965.
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Paez-Valencia, J., Otegui, M.S. (2020). Cell-Free Protein Translation System for Expression of Lipid-Binding Proteins Tagged with Small epitopes and Their Use in Protein–Lipid Overlay Assays. In: Otegui, M. (eds) Plant Endosomes. Methods in Molecular Biology, vol 2177. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0767-1_12
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DOI: https://doi.org/10.1007/978-1-0716-0767-1_12
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Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0766-4
Online ISBN: 978-1-0716-0767-1
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