Versatile tool for generation of ubiquitin-E2 thioesters.

1. Ubiquitinylation of target proteins in presence of dedicated E3 ligase. Panel of E2s provided for generation of E2-Ub thioester conjugates for testing vs. specific E3/target combinations. For example: ubiquitinylation of p53 in the presence of mdm2 (E3) and UbcH5b (E2)10. 2. Activation of Ub for thioester conjugation to novel E2 enzymes (substituted like for like with kit E2s, under directly comparable conditions). 3. Use of cell lysate or crude fractions/preparations as source of E3 ligases to facilitate ubiquitinylation of purified target proteins in the presence of ubiquitinylation kit components. 4. Substrate (target) independent in vitro ubiquitinylation reactions. Determine ubiquitin ligase activity/specificity of proposed E3 enzymes and/or their catalytic domains/fragments11. Note: Protocols provided for applications 1 and 2. Assay set-up can be readily modified for alternative applications by inclusion, omission or substitution of specific enzyme components.

For generation of ubiquitin-E2 thioesters for use in ubiquitinylation experiments. This kit provides the means of generating a range of thioester-linked ubiquitin conjugation enzymes (E2s), utilizing the first two steps in the ubiquitin cascade; for use in the transfer of ubiquitin to E3 ligases; and the subsequent ubiquitinylation of target proteins.Biotinylated ubiquitin provided supports thioester formation and high sensitivity detection of ubiquitin conjugates. Suggested uses: Generation of ubiquitin-E2 thioesters for use in ubiquitinylation experiments, Ubiquitinylation of target proteins in the presence of a dedicated E3 ligase, Activation of ubiquitin for thioester conjugation to novel E2 enzymes, Use of cell lysate or crude fractions/preparations as source of E3 ligases to facilitate ubiquitinylation, Substrate (target) independent in vitro ubiquitinylation reactions.