If I performed coip between e3 ligase and its putative substrate with or without MG132, the substrate band would be enriched by e3 ligase with MG132. So I say that MG132 stabilizes the protein complex. This means the interaction between e3 ligase and substrate will promotes substrate degradation. Supposing that I use similar level of substrate protein for coip with MG132 or without it, after the experiment, the level of substrate will be different. With MG132, the substrate level will be higher. My question is: the enrichment of substrate with MG132 may be only caused by the higher concentration of substrate. So this result can't demonstrate that their interaction can promote substrate degradation. (Substrate may be degraded by other endogenous e3 ligases but not the one of interested) Am I right?
As far as I know, when u do cell lysis, you have to add protease inhibitor cocktail to prevent the degradation of protein. In some cases, when the protein has high turn over rate, we treat cell with MG132. In my opinion, if cells dont degrade proteins, they will not produce more protein so it will not affect the concentration of substrate.
I think you compare co-IP between sample treat or without treat MG132 based on the internal control.
You are correct. All that it shows is that the substrate is sensitive to proteasomal degradation. To show that your E3 is involved in this, you would need to transfect a ligase-activity incapacited mutant E3 (in the hope that this mutant will still bind the substrate with the same affinity as the wild-type, but will not ubiquitinylate it). Be careful in interpretation as the substrate may be uubiquitinylated by more than one E3.
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