Abstract
Ubiquitin ligases (E3s) function by binding to both a protein substrate and to ubiquitin-conjugating enzymes (E2s) bound to ubiquitin. E3s facilitate the transfer of ubiquitin from the E2 active site to an E3-bound substrate. Thus, the affinity of the interaction of an E2 with its E3 partner is of considerable interest. The purpose of this work is to (1) provide protocols for the purification of the human E2 Cdc34, as well as for some additional protein components needed for the assays described here whose purification protocols haven’t been described elsewhere in detail; (2) provide the researcher with critical information regarding the proper long-term storage of these enzymes to retain maximal activity; (3) provide a protocol to benchmark Cdc34 activity with previously described activity levels in the literature; and (4) provide a simple and rapid means of measuring E2 affinity for an E3.
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Acknowledgments
This work, including the efforts of Spencer Hill, Connor Hill, and Gary Kleiger, was funded by HHS | National Institutes of Health (NIH) (R15 GM117555- 01).
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Hill, S., Hill, C., Kleiger, G. (2018). Using In Vitro Ubiquitylation Assays to Estimate the Affinities of Ubiquitin-Conjugating Enzymes for Their Ubiquitin Ligase Partners. In: Mayor, T., Kleiger, G. (eds) The Ubiquitin Proteasome System. Methods in Molecular Biology, vol 1844. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8706-1_4
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