Discrepancy between tissue and cell culture - (Jul/20/2012 )

I am looking for the phosphorylated form of a protein. I have some puzzling results.

When I look for the phosphorylated from directly from the tissue protein extract, it is not there. When I look for it in protein extract of the cell culture grown from the same tissue (although different sample), it is there. How is this possible?

As for total protein (active and inactive), it is present in both tissue and cell culture.

Could it be that phosphatases left behind in the tissue protein extract have removed the majority of the phosphate groups in the tissue but this is not happening in the cell culture tissue?

Both protein extractions and storage are processed in the same way and stored in -80C. I am using a protease inhibitor but not a phosphatase inhibitor.

Thanks!

Cell culture is well known to alter expression of many genes, whether or not this is happening for your sample is very diffcult to say.

However, depending on the tissue, you may well have a mixed population of cells which may well be producing phosphatases that are not present in the more limited population of cultured cells. Try the experiment with phosphatase inhibitors, they should make a difference.