I've been trying to isolate both mitochondria and nuclei from the rat brains that I work with. But when I run blotting some proteins appears in every fraction of the sample, B-actin and COX4 for example appeared in every fraction (nuclei, cytosol and mitochondria) Im not sure if COX4 should have appeared like that. I think something with the protocol Im currently using to isolate the fractions could be the problem. Im using two centrifugations, the first one give me the nuclei (800g / 10 min) and the second the mitochondria (9200g / 15 min). Woul anyone give me some light?
PS: Sorry for english, thats not my first language