I have been trying to repeat an experiment for several weeks now without any success (welcome to science, right?). I previously found that my protein of interest is ubiquitinated and so I treated with MG132 to confirm that it is degraded by the proteasome. I've tried to repeat this experiment several times now and do not see any increase in protein concentration following MG132 treatment.
I've tried ordering new MG132 and treating with lactacystin but still no luck. For my treatment conditions I'm using 10, 20, or 40 uM MG132 or lactacystin for 3 and 6 hours, with or without serum-free medium.
Based on my results from this and other related experiments I seriously doubt that this negative result is due to this protein not actually being degraded. Any thoughts or suggestions? Thanks in advance.