I've protocols for isolation of membrane bound proteins via treatment with ice cold sodium carbonate. If anyone could speak to the efficacy of this vs centrifugation, that would be swell.
I presume your trying to show that an ORF in the mtDNA of your fungus that encodes a 1900aa protein is actually translated? If so, why not just do a mitochondrial translation assay. The basic protocol is to block cytosolic translation with cyclohexamide, and incubate your cells with S35 methionine. Since mitochondrial ribosomes are not inhibited by cycloheximide, mitochondrial proteins will be exclusively S35 labelled, Then you can run a protein gel and look for an S35 labelled protein at the predicted size.
My PI's skepticism also stems from the fact that the translation assay didn't reveal the protein when he did it way back when.
The ultimate goal here is to, on the off chance the protein is visible, to cut it out and send it for ab production.
A while ago I was working on the characterization of protein complexes in the inner mitochondrial membrane from the fungus Podospora anserina. The attached publication by Frank Krause et al. might be interesting for you because it contains BN-PAGE and CN-PAGE protocols optimized for the use with fungal mitochondria.
Regards, Christian
Article Supramolecular Organization of Cytochrome c Oxidase- and Alt...
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