I am incubating bacterial vesicles that contain proteases with human proteins. I want to identify the site of cleavage in these proteins but I would like first to separate the vesicles from 1) uncleaved protein, 2) peptides generated after cleavage. I don't know the cutting sites specifically so I don't know the size of the peptides generated. I want to send the samples to Mass Spectrometry to identify the cutting sites.
I have no experience in the separation of vesicles from protein/peptides. What I did first was to try to ultracentrifuge the OMVs and take the supernatants, but we think that proteins are maybe sticking to the glass or to the vesicles since I can't see any protein left in the supernatant (by Coommassie staining). Maybe I am not using enough protein? DO you any experience in the identification of cleavage sites in proteins?
Thank you
I don't think there is enough protein in there without the EVs to show positive results, maybe try a micro BCA. Also, maybe use a technique that separates particles by size like TFF to isolate the proteins without them being destroyed or sticking to EVs and or tube.
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