I need to pick one spot of my protein of interest, but in DIGE the same protein from different samples is placed in the same position. How can I separate the samples that are superposed in the same position in order to analyze them on MS?
If I understood right, you have the protein N in your samples A and B and you want to pick up only the protein from the sample A (NA), and not the sample B (NB)? Is it right? Do you have enough samples to run another DIGE with only the sample A? Or if not, is there any physical or chemical property that you could use to segregate NA from NB? They have the sample pI and molecular mass, so something different...
Hi,
Why would you want to separate these species? After all, it is the same protein, so the more material you have, to higher the chance you'll be able to identify the protein.. Moreover, depending on the type of labeling (minimal or full), there is insufficient difference between the two (differentially labeled) protein subsets to allow for separation, I guess.
If you suspect that there are differences between the proteins stemming from both samples, like post-translational modifications, this will affect the migration in the IEF and/or PAGE direction, resulting in different spots for the different proteins.
Hope this helps!
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