SM
Shin-ya Miyagishima
  • Department of Cell Genetics, National Institute of Genetics, Japan
Research fields
  • Plant science
Preparation of Chloroplast Lipid Membrane and Lipid-protein Interaction Assay
Authors:  Kumiko Okazaki, Shin-ya Miyagishima and Hajime Wada, date: 02/05/2016, view: 9620, Q&A: 0
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.
Measurement of PI4P Levels in Intact Chloroplasts Isolated from Arabidopsis thaliana
Authors:  Kumiko Okazaki, Shin-ya Miyagishima and Hajime Wada, date: 02/05/2016, view: 9468, Q&A: 1
Phosphatidylinositol 4-phosphate (PI4P), a major species of phosphoinositides, modulates many fundamental cellular processes. We have recently revealed that PI4P plays an important role in chloroplast division as a negative regulator. Despite its importance in chloroplasts, the content of PI4P in chloroplasts is very low and it is difficult to measure PI4P levels. In this protocol, we describe a simple method that we have developed for measurement of low level of PI4P in chloroplasts. Intact chloroplasts were isolated by a basic method using Percoll gradient centrifugation and acidic lipids were extracted from the isolated chloroplasts. The extracted acidic lipids including PI4P were spotted onto the membrane strip, which had been pre-spotted with PI4P standards and other phosphoinositides as negative controls. PI4P in the spot of acidic lipids on the membrane was detected using a PI4P binding protein.
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