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Studying Protein Interactions with Luciferase Reporters

  • Yeast two-hybrid systems use interacting protein domains to bring a DNA-binding domain and a transcriptional activator close enough together to activate a reporter gene.
  • This strategy works in mammalian cells as well.

Here's how it works:

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  • Clone your genes for your "BAIT" and "PREY" proteins into the pBIND and pACT vectors, respectively.
  • Co-transfect your cells with pBIND, pACT and a responsive luciferase vector containing 5 repeats of the GAL4 response element in front of a minimal promoter (e.g., pGL4.31).
  • After culturing the cells, perform a Dual-Luciferase® Reporter assay.
  • If your BAIT and PREY proteins interact, you will see an increase in luciferase activity. The Renilla luciferase control in the pBIND vector allows you to normalize the firefly luciferase activity.

Identifying Transcription Factor Targets of cdk3

The authors of this study proposed that cdk3 may be targeting transcription factors and used the CheckMate™ System to search for targets of cdk3.

Zheng, D. et al. (2008) Cancer Res. 68, 7650–60.

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Transcription Factor Results
E2F1 (– control) basal activity
E2F2 (+ control) >3-fold increase
ATF1 >15-fold increase
ATF2 basal activity
ATF3 basal activity
STAT2 basal activity

Tools

Above downloads in PDF format

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