I´m able to induce somatic embryogenesis with leaves and seeds in the same species.
If I realize the proteome analysis, could i compare the two tissues during the induce process? i.e., two explants analysed in the same induce periods - 7 days. Is it possible to identify common spots?
I believe that I cannot identify the expression's variation from %Vol.
But from common spots, could I identify the proteins related to this process of two tissues by mass spectrometry?
Hi Marilia,
The easiest way to start with is : label your extract (from induced cells) with Cy3 and the other one with Cy5 and then mix your extract and load your mix sample on IPG strips (continue with 2D as usual). You will find green , Red and yellow spots. the yellow spots represent the common proteins (same expression level).
If you want to analys results from both tissues treated and control at the same time then you should run several gel and start with matching the gels to see differences and pick up the spots for identification..
Good Luck
Fluorescent stains are good if you have it as suggested by Bahram - in comassiae staining or silver staining you have to make a incorporate spots from both leaf sample and seed sample in your base gel image for comparison - that can be a little problematic if both the 2D gels do not run a exactly similar pattern - sometimes high abundant proteins like RUBISCO in green tissues interfere a little in the 2nd D and gels may be a little different than that in seeds. Also, it is better if you can do a MALDI of both spots (from leaf and seed sample) as sometimes protein modifications also change the position in the 2nd dim which may be different for leaf and seed.
Hi Marilia
I would change the approach. The risk of your differential proteome analysis between leaves and seeds is to have a huge amount of data, namely lots of differentially expressed spots, and discover, after MS protein identification, that the proteins founds are not involved in embryogenesis. I would suggest to choose some key proteins involved in embryogenesis, immunoprecipitate in non denaturing conditions in both samples, then 2DE and MS analyses to identify proteins differentially (or common) involved in embryogenesis. I think you will have more chances. I hope that I have been useful to you, good luck
Maria Teresa
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