Improve Research Reproducibility A Bio-protocol resource
Concise Method
tooltip

Mitochondria isolation for Western blotting

CF Cécile A. Ferré
AT Anne Thouard
AB Alexandre Bétourné
AD Anne-Louise Le Dorze
PB Pascale Belenguer
MM Marie-Christine Miquel
JP Jean-Michel Peyrin
DG Daniel Gonzalez-Dunia
MS Marion Szelechowski
request Request a Protocol
ask Ask a question
Favorite

We performed sub-cellular fractionation to obtain mitochondria-enriched fractions. To this aim, 2.107 rat primary cortical neurons were pelleted, rinsed twice in PBS and resuspended in homogenization buffer (HB: 10 mM Tris–HCl, pH7.5, 2 mM MgCl2, 10 mM KCl, 250 mM Sucrose, Protease Inhibitor cocktail, 0.5 mM DTT). Neurons were then frozen at – 80 °C and thawed, transferred to a 2 ml Potter homogenizer, triturated for 20 times and further centrifuged at 1,000 g for 10 min. The supernatant was then centrifuged at 12,000 g for 15 min, afterwards the pellet, containing mitochondria, was resuspended in HB + 1% Triton X-100 for protein quantification by Western Blot.

Copyright and License information: The Author(s) ©2021
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

post Post a Question
0 Q&A