The TOR ('target of rapamycin') protein kinase is conserved from yeast to humans and controls cell growth in response to nutrient availability. I am planning to measure TOR activity in extracts prepared from filamentous fungi that are grown under nutrient-rich and starvation conditions. Does anybody have a protocol or suggestions how this could be done? Or would it be possible to adapt a protocol established for measuring TOR activity in baker's yeast or mammals for use in filamentous fungi? Help / hints would be greatly appreciated!
Are you trying to measure the level of the protein or the message?
@Matthew: The determination of TOR protein levels is not so relevant at the moment. I would be more interested whether its kinase activity is changed under various experimental conditions, for example. My basic question is: how active is TOR signaling in my samples that show different levels of autophagy induction? It would also help me if there is a downstream target of TOR signaling that could be assayed conveniently and would provide a clear read-out.
Did you try kinase assay kit like the following?
http://www.lifetechnologies.com/order/catalog/product/PV5099
But it will be good to do the test in presence or absence of a specific inhibitor of TOR.
Do +/- rapamycin. If you can find a downstream target, maybe performing some sort of pulse chase to measure phosphorylation levels of the target with radioactive precursor. Using 32P
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