Isolating ubiquitinated proteins
Updated January 15, 2015
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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.

The following table shows relevant stocks.

Stock # Genotype
Chromosomes
58758 y[1] w[*]; P{w[+mC]=UAS-Ubi6-birA}3 1;3
58759 y[1] w[*]; P{w[+mC]=UAS-birA.F}2 1;2
58760 y[1] w[*]; P{w[+mC]=UAS-birA.F}3 1;3
58982 w[*]; P{w[+mC]=UAS-Ubi6-birA}2; TM2/TM6B, Tb[+] 1;2