Published: Vol 5, Iss 7, Apr 5, 2015 DOI: 10.21769/BioProtoc.1435 Views: 13616
Reviewed by: Fanglian He
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Abstract
Many postitive-stranded RNA viruses, such as Hepatitis C virus (HCV), highjack cellular membranes, including the Golgi, ER, mitchondria, lipid droplets, and utilize them for replication of their RNA genome or assembly of new virions. By investigating how viral proteins associate with cellular membranes we will better understand the roles of cellular membranes in the viral life cycle. Our lab has focused specifically on the role of lipid droplets and lipid-rich membranes in the life cycle of HCV. To analyze the role of lipid-rich membranes in HCV RNA replication, we utilized a membrane flotation assay based on an 10-20-30% iodixanol density gradient developed by Yeaman et al. (2001). This gradient results in a linear increase in density over almost the entire length of the gradient, and membrane particles are separated in the gradient based on their buoyant characteristics. To preserve membranes in the lysate, cells are broken mechanically in a buffer lacking detergent. The cell lysate is loaded on the bottom of the gradient, overlaid with the gradient, and membranes float up as the iodixanol gradient self-generates. The lipid content of membranes and the concentration of associated proteins will determine the separation of different membranes within the gradient. After centrifugation, fractions can be sampled from the top of the gradient and analyzed using standard SDS-PAGE and western blot analysis for proteins of interest.
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Acknowledgments
This work was supported by funds from the Gladstone Institutes, and the National Institutes of Health (R056 AI069090 (MO), and P30 DK026743 (MO) (University of California – San Francisco Liver Center). We gratefully acknowledge support through the Training Grant (T32 DK060414) from the US National Institute of Health to DAV.
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Article Information
Copyright
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Vogt, D. A. and Ott, M. (2015). Membrane Flotation Assay. Bio-protocol 5(7): e1435. DOI: 10.21769/BioProtoc.1435.
Category
Microbiology > Microbe-host interactions > Ex vivo model
Microbiology > Microbial cell biology > Organelle isolation
Cell Biology > Organelle isolation > Fractionation
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