I have used iTRAQ for analysis of the arabidopsis secretome, however, I am now hoping to apply this method to the whole proteome of various crop species.
Advice on sample preparation or alternative proteomic methods would be extremely useful.
Thank you.
Sure, this method may offer a broader view on global effects of miRNA-mediated expression repression (direct as well as indirect). However, since it delivers no information on specific (direct) effects of the miRNA, candidate analyses have to be done to validate or falsify first hints obtained by global analyses.
Hi,
I have recently started working on the Mass Spec data analysis, and I have hard time finding iTRAQ labelled analysis protocols. More and more people are going for unlabelled analysis.
This is not an answer, but a point that, do you have any plan to go for unlabelled analysis? This might be prevalent in future.
Good luck.
Hi Heather,
I am not sure exactly what your method is so I can only give general advice.
I use a heavy N2 labelling system in a method similar to SILAC; This gives me the opportunity to mix samples and then run together and isolate the Heavy from the light for analysis. So if you are want to quantify the affects of the silencing holistically, this may be a option.
If you just want to quantify the silenced transcript, this may be a bit overkill.
qRT PCR should sort that out.
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