Tissue Mito-IP: Immunopurification of Mitochondria from MitoTag Mice

Cell organelles represent a minor fraction of the total cellular content, making whole-cell profiling inadequate for monitoring changes in the mitochondrial proteome, metabolome, and lipidome. Traditional techniques for purifying mitochondria have inherent limitations, often compromising organelle purity, isolation time, or viability. Additionally, the components of conventional organellar isolation buffers, such as sucrose, can interfere with mass spectrometry (MS) profiling. To overcome these challenges, a novel method called 'Mito-IP,' was developed which facilitates the rapid immunopurification of pure and intact mitochondria. This method enables mitochondrial isolation within 10 minutes and supports various downstream applications, including immunoblotting, proteomic, metabolomic, and other -omic analyses. The method employs a chimeric protein comprising of three HA epitope tags fused to the outer mitochondrial membrane protein OMP25 (rat, aa109-145). An LC/MS-compatible buffer (termed 'KPBS') was also developed containing only KCl and KH₂PO₄, significantly improving performance and mitochondrial viability. Following the success of the Mito-IP method, the same epitope-tagged concept is being extended to the isolation of other organelles, including lysosomes (Lyso-IP), Golgi (Golgi-IP), and peroxisomes (Peroxo-IP). The following optimised protocol details the immunoprecipitation of mitochondria from tissues of MitoTag mice. The same steps apply to the immunopurification of other organelles when the HA-epitope tag is present on the organelle of interest.