Metabolically Biotinylated Reporters for Electron Microscopic Imaging of Cytoplasmic Membrane Microdomains

  • Kimberly J. Krager
  • John G. KolandEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1376)


The protein and lipid substituents of cytoplasmic membranes are not in general homogeneously distributed across the membrane surface. Many membrane proteins, including ion channels, receptors, and other signaling molecules, exhibit a profound submicroscopic spatial organization, in some cases clustering in submicron membrane subdomains having a protein and lipid composition distinct from that of the bulk membrane. In the case of membrane-associated signaling molecules, mounting evidence indicates that their nanoscale organization, for example the colocalization of differing signaling molecules in the same membrane microdomains versus their segregation into distinct microdomain species, can significantly impact signal transduction. Biochemical membrane fractionation approaches have been used to characterize membrane subdomains of unique protein and lipid composition, including cholesterol-rich lipid raft structures. However, the intrinsically perturbing nature of fractionation methods makes the interpretation of such characterization subject to question, and indeed the existence and significance of lipid rafts remain controversial. Electron microscopic (EM) imaging of immunogold-labeled proteins in plasma membrane sheets has emerged as a powerful method for visualizing the nanoscale organization and colocalization of membrane proteins, which is not as perturbing of membrane structure as are biochemical approaches. For the purpose of imaging putative lipid raft structures, we recently developed a streamlined EM membrane sheet imaging procedure that employs a unique genetically encoded and metabolically biotinylated reporter that is targeted to membrane inner leaflet lipid rafts. We describe here the principles of this procedure and its application in the imaging of plasma membrane inner leaflet lipid rafts.

Key words

Membrane protein Membrane microdomain Nanodomain Lipid raft Electron microscopy EM imaging Avidin-biotin detection Gold labeling 



The authors wish to acknowledge the University of Iowa Central Microscopy Research Facility for their expert technical support and guidance, and for their provision of instrumentation used in TEM imaging.


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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of PharmacologyThe University of Iowa, Carver College of MedicineIowa CityUSA
  2. 2.Division of Radiation HealthUniversity of Arkansas for Medical Sciences, College of PharmacyLittle RockUSA

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