I want to do tissue subcellular fractionation of mouse liver. According to the method, the speed to separate ER is over 200,000g for 120 min (Song et al., 2006). However, liver exosome isolation only needs 100,000 x g for 70 min (Ishiguro et al., 2019). Thus, I'm wondering how to separate exosome and ER?
Song, Y., Hao, Y., Sun, A., Li, T., Li, W., Guo, L., ... & Wei, H. (2006). Sample preparation project for the subcellular proteome of mouse liver. Proteomics, 6(19), 5269-5277.
Ishiguro, K., Yan, I. K., & Patel, T. (2019). Isolation of tissue extracellular vesicles from the Liver. JoVE (Journal of Visualized Experiments), (150), e58649.
Ishiguro et al did not lyse the cells they just used collagenase to detach cells into a cellular suspension. So, if you are using their method then the ER is inside the intact cells pellets retrieved after 50xg, 300xg, 200xg and 10,000xg, perhaps you can skip directly to 10,000xg. You can isolate the EV fraction from resultant supernatant after 10,000xg spin by ultracentrifugation of supenatant at 100,000xg. Then go back to the cellular pellets, suspend them together and burst the cells open with a homogenizer in homogenization buffer and centrifuge to get ER fraction. Personally, i worked on canine brain and kedney and liver but to get microsomal (ER) pellet i used 110,000xg, 80,000xg, and 80,000xg. Either way the ER is retained in the initial cellular pellets not EV pellet.
Bear in mind that you will still have mitochondrial and nuclear contaminants in the microsomal fraction which sediment at lower velocities than 100,000xg. So start with getting rid of these at their specific speeds and use final supernatant to get ER fraction.
Is your target EVs only? I used collagenase and DNase together with mechanical disruption (cutting) and checked possible contamination by calnexin or RPL5. You can get a purer EV yield by applying a sucrose cushion.
For more information, you can check my published paper
Article Comparison of separation methods for tissue‐derived extracel...
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