Figure 5
From: Accurate estimation of 5-methylcytosine in mammalian mitochondrial DNA
Preparation of mitochondria and highly purified mtDNA. (a) Flow of mitochondrial preparation with three grades of mitochondrial fractions and key experimental steps. (b) Western blot analysis of total cell lysates (total lysates) and nuclease/protease-treated mitochondrial preparations (N-Mito) from mouse livers using antibodies against Fp70, p32 and histone H3. The panel with histone H3* is a longer exposure image of the panel above. (c) An example of ethidium bromide stained image of liver total NA and liver mtNA purified from N-Mito; they were treated with the restriction enzyme EcoRV, which cuts mouse mtDNA twice to generate 9.5 and 6.8 kb fragments. While corresponding bands were not seen in total NA due to the small proportion of mtDNA compared to nuclear DNA, two bands corresponding to the EcoRV-generated mtDNA fragments were observed in mtNA from N-Mito. A DNA ladder (M) was run on the same gel but not next to the sample lanes. Full-length gel and blots are presented in Supplementary Fig. S6c,d. Note that larger amounts of mtNA were treated with EcoRV and RNase T1 and were electrophoresed for gel purification of mtDNA for LC/MS analysis.
