Autophagy flux method? - (Jun/23/2010 )
I have recently started on a new project regarding autophagy on cultured normal human cells (fibroblasts). And since most of the methods describing autophagy are at "fixed" timepoints (E.g. using LC3 as a molecular marker), I would like to know whether autophagy is carried out in the cells the whole way.
So do you know any methods which can describe the efficiency of autophagy, or the autophagic flux?
The optimal method would be: introduce foreign misfolded/aggregated protieins into the cells; induce autophagy (with e.g. starvation); measure the lower concentration of the foreign misfolded/aggregated proteins.
So if autophagy should work propperly, it should clean up some of the "bad" stuff inside the cells, how to measure this?
I hope my questions makes sense (at least for me it does)..
You can use the protein p62 as a marker. Without autophagy the level of this protein is fixed, however when autophagy is induced more and more of this protein will be degraded. Therefore you can compare the level of p62 to detect the autophagic flux.