Chapter title |
Cell Cycle Oscillators
|
---|---|
Chapter number | 18 |
Book title |
Cell Cycle Oscillators
|
Published in |
Methods in molecular biology, January 2016
|
DOI | 10.1007/978-1-4939-2957-3_18 |
Pubmed ID | |
Book ISBNs |
978-1-4939-2956-6, 978-1-4939-2957-3
|
Authors |
Jarvis, Marc A, Brown, Nicholas G, Watson, Edmond R, VanderLinden, Ryan, Schulman, Brenda A, Peters, Jan-Michael, Marc A. Jarvis, Nicholas G. Brown, Edmond R. Watson, Ryan VanderLinden, Brenda A. Schulman, Jan-Michael Peters |
Abstract |
The anaphase-promoting complex/cyclosome (APC/C) is a 1.2 MDa ubiquitin ligase complex with important functions in both proliferating and post-mitotic differentiated cells. In proliferating cells, APC/C controls cell cycle progression by targeting inhibitors of chromosome segregation and mitotic exit for degradation by the 26S proteasome. To understand how APC/C recruits and ubiquitylates its substrate proteins and how these processes are controlled, it is essential to analyze APC/C activity in vitro. In the past, such experiments have been limited by the fact that large quantities of purified APC/C were difficult to obtain and that mutated versions of the APC/C could not be easily generated. In this chapter we review recent advances in generating and purifying recombinant forms of the human APC/C and its co-activators, using methods that are scalable and compatible with mutagenesis. We also describe a method that allows the quantitative analysis of APC/C activity using fluorescently labeled substrate proteins. |
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