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Figure 5 | BMC Cell Biology

Figure 5

From: Protein-trap version 2.1: screening for expressed proteins in mammalian cells based on their localizations.

Figure 5

Synthesis of full-length epitope-tagged proteins. Epitope-tagged ATP5B and ACTB proteins were analyzed by Western blot to confirm the translation of full-length proteins. The Western blots were first probed with anti-myc antibody and secondly with anti-ATP5B or anti-ACTB following the stripping of the anti-myc antibody. Cell lysates before (-Cre) and after (+Cre) the Cre-mediated excision of the SA-IRES-puromycin cassette were analyzed. Specific myc-epitope signal (••) was detected from the (+Cre) cell lysates. No myc-epitope signal was detected from the (-Cre) cell lysates. Anti-ATP5B and anti-ACTB detected both endogenous ATP5B and ACTB proteins (••••), respectively, in addition to the epitope-tagged corresponding proteins (••). Slight size difference (approximately 3–4 kDa) between the epitope-tagged protein (••) and the endogenous counterpart of each protein (••••) reflects the addition of the mini-exon encoded epitope-tag. The "+cre" cell lysates were prepared from the cells that were transiently infected with the cre virus. Thus, they are mixtures of cells that express the tagged proteins and the cells that do not. As the cre-mediated excision efficiency is in the range of 0.5%-2%, the myc-tagged protein bands are always stained less intense. Molecular weight (in kDa) standards are also indicated.

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