Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.
Detection of protein ubiquitination in cultured cells
Detection of ubiquitination on target protein through In vitro ubiquitination assay
8 μl | 5X ubiquitination buffer |
250 ng | ubiquitination E1 |
500 ng | ubiquitination E2 |
0.5 μg | ubiquitin |
0.5 μg | protein of interest |
water to 40 μl total volume |
In this presentation, we first described steps that permit detecting ubiquitin modification on a protein of interest in cultured mammalian cells. In order to detect ubiquitination specifically on the protein of interest, not on non-covalently interacting proteins, we used a stringent condition for cell lysis, immunoprecipitation and washing 1,3. Ubiquitination, detected by immunoprecipitation of target protein in such a harsh condition followed by anti-ubiquitin immunoblotting, is therefore likely to be specific to the target protein. To prevent protein deubiquitination during the experimental procedures, deubiquitinating enzyme inhibitors such as N-ethylmaleimide and ubiquitin aldehyde may be added to all buffers 4. However, it is unlikely that the deubiquitination enzymes remain active after 10 min boiling procedure. Strong smears or ladders of high molecular species are typically the result of ubiquitination. The degree of ubiquitination of the protein of interest can be assessed by comparing the ratio of ubiquitinated/unmodified target protein in several experiment conditions. Meanwhile, free ubiquitin molecules are generally reduced when ubiquitination of target protein is increased. This protocol is also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.
We also described an in vitro ubiquitination assay using purified or recombinant proteins. Various epitope-tagged ubiquitination components are commercially available. Ubiquitination E3 ligase is not necessary for ubiquitin conjugation in vitro.
The authors have nothing to disclose.
This work was supported by NIH grants RO1 DC006497, RO1 NS057289, and PO1 ES016738 (to Z. Zhang); California Institute for Regenerative Medicine grants RS1-00331-1 and RL1-00682-1 (to Z. Zhang); the American Parkinson Disease Association (to Z. Zhang and Y. S. Choo); and the Michael J. Fox Foundation for Parkinson’s Research (Z. Zhang).