I am doing a western of a protein that is encoded in the nucleus but is then imported into the mitochondria. The immature protein contains a mitochondrial targeting sequence that is cleaved upon import. The mature protein is 31 kDa and targeting sequence is 4 kDa. Does anyone know if I would expect a double band on a western? Has anyone seen a double band with a mitochondria protein encoded by nucleus?
If you resolve the proteins well you should be able to see both bands. I would go with a 12% gel and run it at 80v until 20-25kDa is at the bottom of your gel. It will probably also depend on the proportions of cleaved vs un-cleaved, whether or not you can see both bands.
Dear Julie,
I normally work with a mitochondrial protein nuclear encoded, with a mitochondrial leader sequence, so I can answer to you without doubts. Most probably in your wb you'll find only a band ( the mature one) but I don't exclude that you will be able to see both, but it is less possible and it is strictly correlated to your antibody. I agree with Richard when he said that it is a matter of ratio between uncleaved/mature form, and I can say that the processed one is always the most abundant. If you need to see the 35 kD band ( pre-protein ) you can do two different things: 1)to perform a wb on a mitochondrial isolation fraction vs the cytosol fractions. 2) treat the cells ( if you work in vitro) with valinomycin ( or FCCP/CCCP to destroy the mitochondrial membrane potential and to inhibit the import) to enrich the unprocessed form. in the end, I can say that I'm able to see my double band only in overexpression condition.
I hope that it could be useful for you.
regards
Francesco
Another factor to take into account would be the half life of the protein, since it will affect the ratio between both bands. Usually the mature accumulates, while the pool of immature newly synthetized, ad associated to chaperones, is small. So, even existing, it is difficult to detect. For big proteins, the Signal peptide represents only a small percentage of the protein, so in a wb you can not resolve them and both theorical bands could appear as one only.
In my experience, in subcellular fractionation, I only detect the mitochondrial pool of the protein, but as Francesco suggests, an overexpression can increment the non-cleaved form.
Best
Andy
Dear Andy, for sure with the sub cellular fraction we find only the mithochondrial pool of protein but adding in an other wb lane the sample coming from the cytosol fraction we could see the un-cleaved protein. But even in this case it could not be sufficient if the antibody doesn't work perfectly. In fact we have to considerate the dilution of the cytosol fraction. For me the best way to see the putative pre-protein is the import inhibition with valinomycin plus an mg132 treatment to avoid proteasomal degradation. Then, an other important consideration is link with the cleavage of the mts. In fact in some case it is only predicted but then you can't see any band.
- Badges
- Science method