Abstract
Lipid-Protein interaction assay is a method to search lipids, which are bound with proteins in vitro. Since membranes that are spotted with chloroplast lipids such as monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol (SQDG) are not commercially available, we extracted these lipids from cyanobacterial cells and spotted them onto membranes. The prepared membranes could be used for lipid-protein interaction assay.
Keywords: Lipid-protein interaction assay, Lipid extraction, Chloroplast lipid
Materials and Reagents
Equipment
Procedure
Representative data
Figure 3. Representative results of Lipid-Protein Interaction Assay. Each spot on the left (PIP Strip) and middle membrane (Membrane Lipid Strip) contained 100 pmol of lipids. The right membrane contained chloroplast lipids, MGDG, DGDG, SQDG, and PG, and also contained cardiolipin (CL) as the control. The membranes were incubated with 0.5 μg/mL GST-PDV2. The abbreviations used are: LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; PI, phosphatidylinositol; PI3P, phosphatidylinositol 3-phosphate; PI4P, phosphatidylinositol 4-phosphate; PI5P, phosphatidylinositol 5-phosphate; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PI(3, 4)P2, phosphatidylinositol 3, 4-bisphosphate; PI(3, 5)P2, phosphatidylinositol 3, 5-bisphosphate; PI(4, 5)P2, phosphatidylinositol 4, 5-bisphosphate; S1P, sphingosine 1-phosphate; PI(3, 4, 5)P3, phosphatidylinositol 3, 4, 5-triphosphate; PA, phosphatidic acid; PS, phosphatidylserine; TAG, triacylglycerol; DAG, 1, 2-diacylglycerol. The image was reprinted from Reference 2 with permission from The Plant Cell; copyright American Society of Plant Biologists (www.plantcell.org).
Recipes
Acknowledgments
This protocol was adapted from the original work (Okazaki et al., 2015) to provide the detailed procedures. This work was supported by JSPS KAKENHI Grant Number 26840089.
References
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