Isolating ubiquitinated proteins

P{UAS-Ubi6-birA} encodes the E. coli biotin ligase (birA) and six copies of ubiquitin tagged with biotinylation target motifs. When expressed in the pattern of a GAL4 driver, the polyubiquitin is cleaved by deubiquitinating enzymes (DUBs), each ubiquitin is biotinylated, and ubiquitin ligases (E1, E2 & E3) modify proteins with the biotinylated ubiquitins. An avidin-conjugated substrate can then be used to isolate biotinylated and ubiquitinated proteins.

Figure of tagging proteins with biotinylated ubiquitins originating in Franco et al. (2011). A Novel Strategy to Isolate Ubiquitin Conjugates Reveals Wide Role for Ubiquitination during Neural Development. Mol. Cell. Proteomics 10: M110.002188.

 

Methods are described in Franco et al. (2011), A Novel Strategy to Isolate Ubiquitin Conjugates Reveals Wide Role for Ubiquitination during Neural Development. Molecular and Cellular Proteomics 10: M110.002188.

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Related links   Fluorescent proteins   lacZ   auxin-inducible protein degradation