I'm looking at the expression of certain proteins in control and injured brain astrocytes and neurons (isolated with MACS). I can't seem to find differences in expression when I know I should at least for some of the proteins (from literature). We think it might be related to the variation in injury level seen in the model we're using. Anyone have any suggestions on how to correlate protein expression levels to spectrin expression levels (which we're using as a 'marker' for cell death)?
Thanks in advance :)
bonjour, si votre test ne donne pas de résultat et que vous êtes sensé à avoir des différences entre votre contrôle et votre lésé, il faut chercher une méthode plus sensible (qui peut révéler mieux ces différences que vous chercher,) ou chercher d'autres marqueurs ou d'autre test complémentaire à votre essai, ....bon courage.
Since you have tried what is suggested in the literature it appears to me that the % of cells that are injured and undergoing death in your model may be relatively tiny compared to the bulk of the tissue/cells and also variable. This might make it hard to get a good correlation in western with small numbers of samples. Imaging may be a better approach where you can localize the injury, score the dead cells based on markers you already know should work. The trouble you are facing is commonly observed by all of us when working with whole tissue or organ after insults. There is no easy way around it. Let me know if you find a better solution.
Thanks Nadija & Raj. Yes, looks like i'll have to find alternative methods to complement. I do intend to do immunohistochemistry to confirm this in parallel but meanwhile in the hope of extracting as much info as I can from these western blots I might do a correlation analysis between the protein and spectrin levels. It is tricky though as very high spectrin levels might correlate with lower protein expression simply due to increased cell death.. Maybe correlating to a cell marker instead would make more sense - i.e. say GFAP for astrocytes and NeuN for neurons or so forth. Every method has its limitations but I guess like u suggests, using an alternative method in parallel is the only answer.
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