In the literature I mostly see western blots for the mitochondrial fission receptor protein Fis1 done on whole cell lysates, rather than on isolated mitochondria. I was wondering why this is the case, as Fis1 is mitochondrial protein, and if it would be appropriate to western blot for Fis1 in the mitochondria fraction alone to increase signal.
Nicole: The short answer to your question is "No need" to use mitochondrial fraction for Fis1 detection with WB, as antibody to which is specific. The best result in practice is to use an HRP-conjugated primary antibody against Fis1, rather than the traditional secondary anti-IgG-HRP which causes many non-specific bands around 50 kDa. Good luck.
Nicole: The short answer to your question is "No need" to use mitochondrial fraction for Fis1 detection with WB, as antibody to which is specific. The best result in practice is to use an HRP-conjugated primary antibody against Fis1, rather than the traditional secondary anti-IgG-HRP which causes many non-specific bands around 50 kDa. Good luck.
Hi Nicole,
it is acceptable (and maybe even recommended) to use mitochondrial protein fractions for Fis1 detection. In fact, there are quite a few papers in which this was shown:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4104696/
http://jcs.biologists.org/content/117/7/1201
etc.
Keep in mind that in mammals the main receptor for recruiting is Mff and not Fis1. Fis1 plays a secondary role in mitochondrial fission (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4666140/).
Cheers, Christian
Hi,
You can see the difference between Western blotting of mitochondrial proteins (Pim-1(UUG) isoform) using total lysates, mitochondrial lysates and mitochondria depleted cytosol in Figure 3A of my paper. Choose the method as you need. Good luck..!!
Article Smac Mimetic with TNF- Targets Pim-1 Isoforms and Reactive O...
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