I can detect my protein (cytosolic soluble of around 35 kDa) by Western Blot of whole cell extracts from rat hearts (adult and neonatal), but when I make lysates from freshly isolated cells the signal is not there anymore.
Can I add protease/phosphatase inhibitors to the digestion buffer?
if you are using enzymatic digestion (collagenase, trypsin, liberase) to isolate single cell suspensions, then yes that can influence your ability to detect this protein if it is cleaved or degraded by the residual activity of the enzymes. Also, cardiac tissue contains cardiomyocytes, fibroblasts, endothelial cells of the vasculature and the surrounding smooth muscle cells. Are you sure that you are isolating the right cell type? Do you plate the cells on plastic and let them grow first? If so , that can also change expression.
if you are using enzymatic digestion (collagenase, trypsin, liberase) to isolate single cell suspensions, then yes that can influence your ability to detect this protein if it is cleaved or degraded by the residual activity of the enzymes. Also, cardiac tissue contains cardiomyocytes, fibroblasts, endothelial cells of the vasculature and the surrounding smooth muscle cells. Are you sure that you are isolating the right cell type? Do you plate the cells on plastic and let them grow first? If so , that can also change expression.
Thanks for replying. Yes I am using collagenase and pancreatin. I lysed the cells with RIPA as soon as the digestion finished. They are primarly beating cardiomyocytes as seen under the microscope. I haven't checked for cultured cells yet. Is there any way to prevent residual enzyme activity during the isolation?
Josh sounds like he knows what he is talking about. Follow his protocol. One last thing that might be an issue although it is less likely. If your protein is associated with cell-cell or cell matrix contacts, such as with adherent junctions or gap junctions or linked to integrins, then dissociating the cells may actually destabilize this cytoplasmic protein. Such a phenomenon may be difficult to deal with experimentally, but it can also tell you something about function.
One more thing (yes there is always one more thing) are you using an antibody against a phosphorylated form? If so, that can change dramatically with dissociation.
well you may get degradation through enzymatic isolation, but the cells should still be making mRNA. Easy to test. But as has been mentioned, cultured cells routinely lose expression of certain proteins and it may not be through the lack of a particular stimulating factor. Cardiac cells don't proliferate much in vivo, i assume, therefore forcing them to proliferate in vitro can have dramatic consequences, an increase in methylation-dependent silencing being one that leaps out straight away. Maybe look at the meth status of your gene if none of the suggested protocols help?
Thank you everyone. Im planning to apply your advice during the next week or so. Ill let you know the results.
I know it has been a while but here it goes the update. I added EZblock (universal protease and phosphatase inhibitors) to the digestion buffer and it did the trick on freshly isolated cells that I checked by immunoblotting. However, I don't seem to keep it after 1 to 2 days of culture. Im working on getting the primers validated to check for mRNA expression.
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