Add like
Add dislike
Add to saved papers

Dual-color pulse-chase ubiquitination assays to simultaneously monitor substrate priming and extension.

Many fundamental discoveries in ubiquitin-proteasome research have relied on reconstitution of activities from purified or recombinantly expressed components. These include landmark discoveries of E1-E2-E3 mechanisms, in which ubiquitin (UB) is initially activated and then covalently shuttled between enzyme active sites and ultimately ligated to substrate or substrate-linked UBs during polyubiquitination. However, recent studies have unearthed enormous variations on the E1-E2-E3 theme; for example, one E3 may employ two distinct E2s, or two different E3s may act in a single assembly or in series, to prime substrates directly with UB and subsequently decorate them with myriad types of polyubiquitin chains. To dissect this complexity, it can be helpful to monitor specific UB transfer reactions in isolation, rather than the end-point products formed upon mixing all enzymes in a cascade. Pulse-chase assays enable observation of a single reaction step and also allow one to differentially label UBs carried by different enzymes within the same tube. In such assays, the "pulse" reaction generates a thioester-linked enzyme~UB intermediate, while the "chase" monitors UB transfer to downstream components over time. Here, we describe pulse-chase assays for detecting fluorescent-UB in E2~UB intermediates. These assays enable direct assessment of particular ligation reactions, alone and in combination, to explore roles of multiple enzymatic cascades in the same tube. We anticipate this technique can be adapted to many different E2s, as well as thioester-forming E3s, to dissect ubiquitination by many distinct enzyme cascades.

Full text links

We have located links that may give you full text access.
Can't access the paper?
Try logging in through your university/institutional subscription. For a smoother one-click institutional access experience, please use our mobile app.

For the best experience, use the Read mobile app

Mobile app image

Get seemless 1-tap access through your institution/university

For the best experience, use the Read mobile app

All material on this website is protected by copyright, Copyright © 1994-2024 by WebMD LLC.
This website also contains material copyrighted by 3rd parties.

By using this service, you agree to our terms of use and privacy policy.

Your Privacy Choices Toggle icon

You can now claim free CME credits for this literature searchClaim now

Get seemless 1-tap access through your institution/university

For the best experience, use the Read mobile app