I am doing in-vitro ubiquitylation reactions where I am treating with DUB's to cleave di-ubiquitin and tri-ubiquitin, but surprisingly level of free ubiquitin is also degrading instead the fact that it should be increased after cleaving of di-ubiquitin and tri-ubiquitin. I need possible explanation for degradation of free ubiquitin.
Koraljka Husnjak
Hi,
how do you detect the level of monoubiquitin after DUB cleavage?
If you detect it by western blot, it might be that monoUb is partially lost because it is small, and does not get retained on the nitrocellulose membrane (if you have 0.45 um membrane).
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