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US 2009/0269731 A1Published application
E3-independent ubiquitinylation assay
AssigneeBurnham Institute for Medical Research
InventorJohn C. Reed
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Abstract
Disclosed herein are compositions and methods for assaying ubiquitination independent of ubiquitin ligase (E3) or a target protein.
Claims11
A method of identifying a ubiquitination modulator comprising
- (a) combining, under conditions that favor ubiquitination activity ubiquitin, a candidate modulator, ubiquitin activating enzyme (E1) and ubiquitin conjugating enzyme (E2), thereby producing a reaction mixture, and
- (b) measuring the amount of polyubiquitin, whereby a difference in polyubiquitin as compared with a reaction performed in the absence of the candidate modulator indicates that the candidate is a ubiquitination modulator.
A method of identifying a ubiquitination modulator comprising:
- (a) combining, under conditions that favor ubiquitination activity:
- (i) tag1-ubiquitin,
- (ii) tag2-ubiquitin,
- (iii) a candidate modulator,
- (iv) ubiquitin activating enzyme (E1), and
- (v) ubiquitin conjugating enzyme (E2), thereby producing a reaction mixture;
- and
- (b) measuring the amount of tag1-ubiquitin bound to said tag2-ubiquitin in said reaction mixture, whereby a difference in bound ubiquitin as compared with a reaction performed in the absence of the candidate modulator indicates that the candidate is a ubiquitination modulator.
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