Try blocking with proteasomal inhibitors. There are plenty to choose from. Methyl ubiqutin is a good start. Most companies carry specific proteasomal inhibitors.

Alternatively do a double immuno precipitation. First with your ab against the protein of interest followed by ubiquitin specific antibody if your protein gets canned by the proteasomal route.

Dear Rajesh, thank you for the answer.

We tryed to use MG132 and Lactocystin (both proteasome specific), but it seems that they are not effecting the degradation significantly. So perhaps it is not a proteasomal degradation. Do you know other inhibitors we can try to clarify what protease is involved?

Alexei, the alternate mechanism could be lysosomal degradation and I say this with caution. Do you have any information regarding your protein of interest i.e. half life of the protein, cellular localization wether it resides on any membrane (possibly exocytosed). One easy way is to take the cell culture supernatant, centrifuge it, take a sample avoiding the pellet and run it on a western blot and see if you can pick up a signal.

Frankly we have a chimera-protein part of it is a GFP, so we can see it in the cytoplasm, and the second part is a part of a rapidly degrading protein which we discovered is hydrolized by the proteasome. However the visible accumulation of this chimera is much lower then the wt GFP. The same in WB. As I wrote previously MG and Lactcys has almost no effect. The amount of mRNA of the wt GFP and our chimera is close, so it is likely not the RNA stability. The half-life we are now estimating using cycloheximide chase. Right now I'm looking for an appropriate protease inhibitor set to find out what may be involved, do you have any idea what would be better to try.

Hi Alexei,

Have you tried Epoxomicin and Bortezomib. Both of them are very specific proteasome inhibitors.

Hi Devi,

No we did not. That time I tryed a couple of proteasome inhibitors and was pretty sure that it was not a proteasomal hydrolysis. I was looking for a good set of protease inhibitors to investigate the hydrolysing activity not related to the proteasome. In fact, we investigated the half-life of the chimera and that was finally sufficent for us.