Hello,

One possibility could be differential centrifugation, where you centrifuge the tissue homogenate/sample first increasing speed (start with 2000g, keep the sediment, take the supernatant and centrifuge at 5000g, keep the sediment, take the supernatant again and spin at 10000g and so on). This way will be able to separate endosomes in the cytosol from those attached/interacting with mitochondria.

Alternatively density centrifugation (sucrose density) could be a possibility, If you have enough isolated mitochondria (the more purified the better), subject them to osmotic swelling and shrinking, sonicate them shortly to break the mitochondria up into vesicles and layer the mitochondrial solution on a sucrose gradient. Centrifuge, divide the sucrose gradient into fractions and analyze them for your protein of interest. Vesicles consisting of inner mitochondrial membrane will penetrate the sucrose gradient the farthest, while vesicles consisting mostly of outer mitochondrial membrane will remain at the top of the gradient.

Good luck with your experiment!

Thank you so much

Following differential centrifugation as described above you can use specific antibody markers for the endosomes (early, late, recycling) and mitochondria to check for contamination/enrichment. EEA1, Rab 5, Rab 7, Rab 11 and a good mitochondrial marker like citrate synthase or TOM20