Has anyone used the following assays to measure ubiquitin activity? If so, please let me know what you think.
1.) Enzo Life Sci (Ubiquitin activating kit, BML-UW0400A-0001)
(or)
2.) Boston Biochem (MuRF1/S5a Ubiquitination Kit, Catalog #: K-102)
(or)
3.) Life Sensors (E3LITE Customizable Ubiquitin Ligase Kit,
Catalog #: UC101).
I believe all these kits are quite good - we have worked directly with both Boston Biochem and Life Sensors. Understanding the experiments is always helpful in making a final recommendation, but all three kits and companies are first rate in their Ubi supplies.
We work routinely with Boston Biochem (Enzo’s) enzymes for our ubiquitination assays, they work well.
We never got them as kit, but we have used the individual enzymes.
We have also tested their His-MuRF1 protein and it was active.
As George mentioned, knowing more about the experiments is important when making a recommendtion for the kit/assay.
MuRF1/S5a Ubiquitination Kit (K-102) from R&D Systems is designed for the in vitro MuRF1-mediated ubiquitination of known substrates and the read-out is based on Western blot using antibodies specific for the target protein.
Ubiquitin Activating Kit Catalog (BML-UW0400A) from Enzo is more versatile and is used to evaluate ubiquitin activation by E1, activity of your E3 ligase utilizing Western blot with Ubi antibody as a read-out after 1-4 h incubation at 37oC.
The E3LITE Customizable Ubiquitin Ligase Kit from LifeSensors (UC101) is basically a "sandwich" ELISA-like highly sensitive assay that relies on enhanced chemiluminescence to quantitatively measure ubiquitin E2 conjugation and E3 ligase activity. It does not require additional non-native tagging or labeling of ubiquitin. The assay is based on the ability of an ubiquitin binding domain to preferentially bind polyubiquitin relative to monoubiquitin, thus measuring activity of E3 ligase that builds ubiquitin chains in a dose-dependent manner. Also, it utilizes 96- (removable) or 384-well (standard) microtiter plate strips well designed for HTS.
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