I am running western blots on liver and hippocampi from high-fat diet mice and normal diet mice. I would like to investigate whether cytochrome c oxidase (CCOX), a marker of metabolism, is reduced in the hippocampus of HFD mice as the literature suggests that this would be the case.
I am assuming that I need to do the same in the liver for it to be a valid experiment? i.e. used an antibody for CCOX both in the liver and hippocampus.
I have never done westerns before and I am unclear on the procedure: if I test for a specific protein in the brain then I guess I automatically need to do the same for the liver?
Thanks in advance.
Irina
Hey Irina,
that depends on the protein you want to investigate. If your protein of interest is only expressed in brain tissue, then you would expect that in the liver tissue this protein is not detectable. In this case, your liver samples are negative controls.
If your protein of interest is expressed throughout the body and you want, for example, check protein expression under different conditions (WT vs KO, different in vivo treatments, High-fat vs low-fat diet), you still do not have to check every organ. You should only investigate organs that are relevant to your questions. But liver (as a metabolic highly active organ) is a good choice.
Since COX is expressed in mitochondria (so nearly everywhere), I would suggest you load your liver and hippocampal samples on one gel and one blot and stain with your Anti-COX antibody. Then you get an overview how this protein changes in two different tissues in dependency of your diet on one blot.
I hope that helps,
All the best,
Marc
IF CCOX level is reduced in case of hippocampus of HFD mice. then you can compare CCOX level in case of hippocampus of other mice model using same detection antibody. you can further establish your finding by observing reduced level of phospholipases or increased level of lipoxygenases by using corresponding antibodies in the western blot experiments.