I am working on a transcription factor which is involved in the ubiquitination pathway and acts as E3 ligase. I have to perform an in-vitro ubiquitination assay to conform the ubiquitination function. I have cloned my orf in pET expression system and successfully purified under denaturing conditions, but could not able to purify under native conditions. I wanted to know whether I can use the protein purified under denaturing conditions for in-vitro ubiquitination assay. Also I would like to know the solvent for E1 and E2 enzymes which are provided in lyophilised form.
You could try to refold your denatured protein. This is more likely to be successful with a small protein than a large one. Basically, refolding consists of gradually removing the denaturant from a dilute solution of the protein under suitable redox conditions.
It may or may not work. You need to have a positive control for ubiquitination activity. The easiest method is to perform an anti-ubiquitin western blot. Your positive control reaction should contain ubiquitin, ATP, a generic E1-E2 pair that you know for a fact is functional, and an E3 (for example, Arabidopsis LOG2 or human MDM2). If the reaction works, you will see a "ladder" of anti-ubiquitin immunoreactivity (exemplary of multiple sizes and types of Ubiquitin-ubiquitin conjugates). As a negative control, leave out the E2 protein. In this lane, you should only see ubiquitin monomers and dimers (no ladder). If your protein of interest is active, it will also form a ladder in the presence of all other components, but not in the absence of E2.
A good buffer system for ubiquitination assays is 50mM Tris + 10mM MgCl2 (pH 7.5) + 1mM ATP + 0.2mM DTT + 10mM Phosphocreatine
Check one of my publication you probably will find what you need .
http://www.ncbi.nlm.nih.gov/pubmed/12769844
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