Abstract
Outer membrane vesicles (OMVs) are spherical bilayered phospholipids of 20-200 nm in size produced from all Gram-negative bacteria and Gram-positive bacteria investigated to date. Previous biochemical and proteomic studies have revealed that the Gram-negative bacteria-derived OMVs are composed of various components like outer membrane proteins, lipopolysaccharides, outer membrane lipids, periplasmic proteins, DNA, and RNA. Here, in this protocol, we describe the method to isolate the OMVs from the culture supernatant of Escherichia coli (E. coli).
Keywords: Outer membrane vesicles, Bacterial extracellular vesicles, Vesicle isolation
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
This protocol was adapted from previously published work (Kim et al., 2013). This work was supported by a grant from the Korean Ministry of Education, Science and Technology, FPR08B1-240 of the 21C Frontier Functional Proteomics Program and Mid-career Researcher Program of National Research Foundation of Korea (NRF) grant funded by the Korea government MEST (No. 20110000215 and No. 20120005634).
References
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For preparation OMVs, I used disposable bottle top filter [Corning® bottle-top vacuum filters, cellulose acetate membrane, sterile, pore size 0.45 μm, membrane diam. 70 mm, funnel capacity 500 mL, case of 12, CLS430514-12EA]
According to our previous data on the stability of OMVs in different conditions, both -80 and 4 C showed similar stability. But I would recommend -80 C for long storage. To check the OMV integrity, I recommend analyzing the size of OMV and shape using dynamic light scattering or TEM imaging.