The homologue of the protein of my interest (POI) is short lived in yeast and the E3 ligase that targets it for degradation is also known. In case of mammalian system testing the half life of POI via cyclohexamide chase assays (CHX) has been a bit challenging due to certain technical limitations. Therefore, I wanted to take an in-vitro ubiquitination approach, at least to get an idea whether or not my POI specific E3 ligase exists in mammalian cells.
Previous articles show that rabbit reticulocyte lysate has been extensively used to provide E1-E2-E3 enzymes for ubiquitination reaction however, I wanted to know if I could utilize lysate prepared from HEK293T cells?
The short answer is Yes. However, it would be challenging to assign specificity to a particular enzyme (because of possibility that an endogenous enzyme may be active). In rabbit reticulocyte lysate one has a better control on the activity, thus specificity.
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