RRC ID 73456
Author Miyata Y, Segawa K.
Title Protocol to analyze lipid asymmetry in the plasma membrane.
Journal STAR Protoc
Abstract The plasma membrane containing cholesterol exhibits phospholipid asymmetry, with phosphatidylcholine and sphingomyelin enriched in its outer leaflet and phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn) on the cytoplasmic side. We herein describe steps for bacterial expression of recombinant proteins that bind to membrane lipids, followed by affinity purification. Using fluorescence-labeled phospholipid analogs, we further detail the assay to detect flippase activity, which maintains the single-sided distribution of PtdSer and PtdEtn, in mammalian cells. For complete details on the use and execution of this protocol, please refer to Segawa et al. (2021).1.
Volume 3(4)
Pages 101870
Published 2022-12-16
DOI 10.1016/j.xpro.2022.101870
PII S2666-1667(22)00750-X
PMID 36595929
PMC PMC9692065
MeSH Animals Biological Transport Cell Membrane / metabolism Mammals / metabolism Phosphatidylcholines* / metabolism Phospholipids* / metabolism
DNA material pQE30/His6-mRFP-NT-Lys (RDB13960) pET28/His6-mCherry-D4 (RDB14300)