Detecting ubiquitylation

Ubitest Flow chart – LifeSensors at tebu-bio.com

Determining the linkage of polyubiquitin on target proteins is challenging. The traditional methods are either through Mass Spectrometry or immunoblot with linkage specific antibodies, which are cumbersome. LifeSensors has developed the UbiTest assay,  a more definitive method for demonstrating the ubiquitylation linkage of a protein, which is to couple immunoprecipitation of polyubiquitylated protein with digestion by a linkage specific deubiquitylase prior to immunoblot analysis. An increased signal for the unmodified substrate or a decreased signal of polyubiquitylated substrate at high molecular weight after K48/K63 specific DUB treatment is a clear indication that the protein was K48/K63 ubiquitylated.

A ready-to-use E3 ligase activity enzymatic assay, the E3LITE kit, has been recently designed enabling researchers to screen for E3 enzyme inhibitors and potentially new therapeutical drugs. What’s it for and why is it of interest?

Traditional methods to detect and purify poly-ubiquitinated proteins require either anti-ubiquitin antibodies or Ubiquitin Binding Associated domains (UBAs). Most of the current methods display rather low affinity for Ubiquitin (UBL) and show only little (if any) specificity for specific ubiquitin linkages (e.g. K63 or K48). To overcome these hurdles, Tandem Ubiquitin Binding Entities (TUBEs) have been designed.