After the identification of numerous proteins to be deregulated in a diseased tissue using iTRAQ what should be the criteria to select a few proteins for their validation using Western blotting.
Hi,
Given the progress in analytical technologies (specifically in quantitative MS methods) over the past decade, I think it is typical that people still want/demand cross-validation of detected changes by Western blotting, which only in a limited number of cases is actually orthogonal to findings obtained by using quantitative MS.
Nonetheless, I don't think you should select candidates necessarily on whether they have been associated to a disease or not: if you find a significantly differing protein that seems unrelated with a disease state, that might even be more interesting..
Down regulation of a protein can have as much impact as up regulation and is therefore equally interesting, so don't hesitate to select a protein that you find to be down regulated in disease!
Even though I think monoclonal antibodies are preferred, it can be quite challenging to find an antibody to your protein(s) of interest at all, so if you do accept a polyclonal.
I think in general with such an approach, you should consider that changes observed by MS can link to specific isoforms or post-translationally modified proteins and these are hardly even picked up by WB (apart from the other intrinsic differences between the two methods!). So if you're unable to confirm your MS-based findings by WB, these could still very well be interesting.
Good luck!
1) Choosing proteins for validation from proteomics data always depends upon the question you have asked. GO analysis of the differentially expressed proteins will help you to figure out the pathways altered and thus can be used to build a hypothesis for your work. You can further go on and choose some of the key proteins of the pathway to validate your data.
2) As said by Dr. Dirksen both up-regulated and Down-regulated proteins are equally important.
4 ) Choosing an uncharacterized protein again depends upon your question but undoubtedly may be important. Characterizing this protein or even reporting an association of an uncharacterized protein with a disease will be addition to the scientific field.
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