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Fig. 5 | BMC Biology

Fig. 5

From: A tissue-specific protein purification approach in Caenorhabditis elegans identifies novel interaction partners of DLG-1/Discs large

Fig. 5

In vivo biotinylation of GTA-tagged protein and removal of endogenously biotinylated proteins by Tobacco etch virus (TEV) cleavage. a Western blot detection of biotinylated proteins in lysed wild-type N2 animals (left lane) and lysed animals expressing GTA-tagged DLG-1 and ubiquitously expressed BirA (right lane). An additional band of the correct estimated molecular weight for DLG-1::GTA is detected upon expression of BirA (arrow). b Western blot analysis of eluate and beads after purification and release of bound bait protein by TEV cleavage. Both blots contain the same samples. The background of naturally biotinylated proteins remains bound to the streptavidin beads and is visible on the α-biotin western blot in the beads remainder, while tagged DLG-1 is cleaved off and visible on the α-GFP western blot in the eluate. No DLG-1 protein is purified in samples from animals not expressing BirA. c Lanes 1 and 2: Western blot analysis of the levels of GTA::CDC-42 purified with GFP-Trap beads (lane 1) and streptavidin beads (lane 2) from animals expressing BirA in seam and hyp7 epidermal cells. Lanes 3 and 4: Western blot analysis of beads (lane 3) and eluate (lane 4) after cleavage of GTA::CDC-42 purified with streptavidin beads. d Schematic of the TEV cleavage procedure

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