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Parkin is an E3 ubiquitin ligase that ubiquitinates multiple substrate proteins on damaged mitochondria, tagging them for clearance via mitophagy. USP30, a deubiquitinating enzyme, antagonizes Parkin-mediated mitophagy by removing ubiquitin from damaged mitochondria. Ubiquitin chains on damaged mitochondria can be phosphorylated by PINK1 are and thought to act as Parkin receptors. Defective mitophagy has been linked to Parkinson’s disease, highlighting the importance of this cellular process. R&D Systems is proud to offer high-quality Boston Biochem products to aid your Parkinson’s disease research.
Antagonize Parkin-Mediated Mitophagy with the Most Active USP30 Enzyme on the Market
The Ubiquitin hydrolysis activity of our USP30 enzyme is significantly higher than that of a competitor’s enzyme.
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Measurement of USP30-dependent Hydrolysis of Ubiquitin AMC. Hydrolysis reactions containing Boston Biochem USP30 (Catalog # E-582) or competitor USP30, Hepes (pH 8.0), 0.1 mg/ml BSA, and 1 µM Recombinant Human Ubiquitin AMC (Catalog # U-550) were carried out in duplicate at 37° C.
Detect Phosphorylated Ubiquitin with Our New Phospho-Ubiquitin (S65) Antibody
Our antibody detects mono- and poly-Ubiquitin chains that are phosphorylated on serine 65 (see data below). It does not cross-react with non-phosphorylated Ubiquitin, Ubiquitin phosphorylated on serine 57 or tyrosine 59, or phosphorylated Parkin.
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Detection of Phospho-Ubiquitin (S65) by Western Blot. Tetraubiquitin chains of each indicated linkage type were incubated for 0-4 hours in reactions containing recombinant PINK1 Kinase (Cat# AP-180) and ATP. At indicated times a portion of each reaction was removed and terminated with SDS-PAGE sample buffer. SDS-PAGE gels (10-20%) were used to resolve approximately 150 ng of Ubiquitin tetramer from each reaction. Western blots were developed using either Rabbit Anti-Human Phospho-Ubiquitin (S65) Affinity-Purified Polyclonal Antibody (Catalog # A-110; upper panels) or Mouse Anti-Human Ubiquitin Monoclonal Antibody (Catalog # MAB701; lower panels). |