I could not detect my target endogenous protein but I can detect over-expressed protein. I used Protease Inhibitor Cocktail Tablets. My friend told me that my target protein could have a high turn over rate so I could not detect the endogenous protein. He recommended me to use MG132 proteasome inhibitor.
1.Should I use both MG132 proteasome inhibitor and Protease Inhibitor Cocktail Tablets to do westernblot or I just use one of both every time I do western blot?
2.Protease Inhibitor Cocktail Tablets and MG132 proteasome inhibitor are used to inhibit protein degradation in western blot. Which one is better?
Thanks
Hi there,
Proteasome is a cellular factory dedicated to the destruction of specifically targeted proteins whereas cellular proteases exert their activity towards possibly any protein, especially during the cell lysis process and in cell extract. So addition of protease inhibitor cocktail is considered systematically when cellular extracts are made. If your favorite protein is a substrate for proteasome, I would advise to further add proteasome inhibitor in your lysis buffer. These inhibitors are used to protect protein during protein sample preparation, they are not specifically used for WB... So use both of them!
Hi there,
Proteasome is a cellular factory dedicated to the destruction of specifically targeted proteins whereas cellular proteases exert their activity towards possibly any protein, especially during the cell lysis process and in cell extract. So addition of protease inhibitor cocktail is considered systematically when cellular extracts are made. If your favorite protein is a substrate for proteasome, I would advise to further add proteasome inhibitor in your lysis buffer. These inhibitors are used to protect protein during protein sample preparation, they are not specifically used for WB... So use both of them!
Hi Nguyen. Just to add to Dominique's answer, you could treat the cells with membrane permeable proteasome inhibitors (like MG123) for ~1-2 hours before doing cell lysis with the inhibitor cocktail. Compare it to cell extract made with inhibitor cocktail, but without MG123.
If your protein is turned over by proteasomes, then you should see more of the protein in MG123 treated cells.
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