I'm new to detection of ubquitinylted (Ub) proteins. I'm going to use a kit called Ubi-Qapture-Q kit by Enzo but I'm not sure how I can detect the Ub-protein by western.
This kit isolates ubiquitnlated proteins from cell extract using a high-binding affinity matrix.
1. When I run the isolated Ub-protein on western gel, I expect to see a smear. The kit says we can use the kit provided Ub-conjugate specific HRP-linked antibody or we can use antibodies to specific protein of interests.
My question is: isn't it worthwhile to detect the Ub-protein by using antibodies specific to protein of interests. This way we can tell if our protein of interest is ubquitinated or not?. What's the purpose of detecting the Ub-protein with the Ub-conjugate specific HRP-linked antibody that comes with the kit?
Also, should we treat our samples with MG132 (proteosome inhibitor) first prior to isolating the Ub-protein by the kit?