Greetings,

Recently, I performed a Cycloheximide chase assay to assess the stability of transiently transfected proteins in HEK293 cells. The CHX chase assay was conducted for 12 hours with following ranges : 0 hour - 1 hour - 2 hours - 4 hours - 8 hours and 12 hours. I tested a control protein (wild type) and a mutant version of that protein, both tagged. For the blot, each well was charged with 25µg of protein. Cycloheximide was used at 50µg per mL in the cell culture media.

For the control protein, my blot was just fine. It is a quite stable protein so I observed no variations in protein quantity over 12 hours, which is the expected result.

However, for the mutant protein, I observed a diminution of the protein quantity for the first 2 hours, followed by a strict further increase for 4, 8 and 12 hours.

So, what could explain such a change ? The CHX I used was quite old so would it be possible that in was degraded or unstable after a while, allowing protein neosynthesis ? Maybe it was not concentrated enough ?

Thanks for your help !