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pNLCoI1[luc2-P2A-NlucP/Hygro] Vector

by Promega
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  • Two Reporter Genes, One Transcript

    Luciferase-based reporter-gene assays remain a useful and powerful method of high-throughput compound screening. However, false hits that result from direct interaction of compounds with the luciferase reporter can result in unnecessary follow-up efforts. The pNLCoI Vectors comprise a second-generation coincidence reporter vector system that allow expression of both firefly luciferase (luc2) and NanoLuc® Luciferase fused to a PEST destabilization domain (NlucP) from the same mRNA transcript. The stoichiometric expression of both luciferases is achieved by use of the P2A sequence from porcine teschovirus-1, which promotes a ribosomal skip and expression of the two unfused enzymes with distinct compound interaction profiles. When used in high-throughput compound screening, false hits caused by direct interaction with one or the other luciferases can be distinguished from true hits that show a similar response for both, reducing workload associated with follow-up screens.

    The pNLCoI Vectors are designed for use with the Nano-Glo® Dual-Luciferase® Reporter (NanoDLR™) Assay System, which sequentially detects firefly and NanoLuc® Luciferase in activity in the same sample with a convenient “add-read-add-read” homogeneous format. Both NanoDLR™ reagents provide stable glow-type luminescence signals with half-lives of approximately two hours for automated batch processing of samples and amenable to assays or high-throughput screens in 96-, 384- or 1,536-well plate formats. Potent inhibition of firefly luciferase coupled with the high-intensity luminescence of NanoLuc® luciferase maximizes sensitivity for detection of both reporters.

    Coincidence reporter assay principle.
    Coincidence reporter assay principle.

    pNLCoI reporter vectors express the firefly luciferase and the destabilized NanoLuc® luciferase reporters on the same mRNA transcript. The two reporters are separated by a short P2A peptide, which causes ribosome skipping and the generation of two soluble, unfused forms of luciferase with distinct inhibitor profiles.

    12456MA-W

    pNLCoI1[luc2-P2A-NlucP/Hygro] Vector GenBank® Accession Number KM359771 and vector sequence text file.

    12458MA-W

    pNLCoI2[luc2-P2A-NlucP/minP/Hygro] Vector GenBank® Accession Number KM359772 and vector sequence text file.

    12457MA-W

    pNLCoI3[luc2-P2A-NlucP/CMV/Hygro] Vector GenBank® Accession Number KM359773 and vector sequence text file

    12459MA-W

    pNLCol4[luc2-P2A-NlucP/PGK/Hygro] Vector  GenBank® Accession Number KM376434 and vector sequence text file.

  • Protocols

    Complete Protocol

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    pNLCoI1[luc2-P2A-NlucP/Hygro] Vector

    PDF (303 KB)

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    pNLCoI2[luc2-P2A-NlucP/minP/Hygro] Vector

    PDF (314 KB)

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    pNLCoI3[luc2-P2A-NlucP/CMV/Hygro] Vector

    PDF (237 KB)

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    pNLCoI4[luc2-P2A-NlucP/PGK/Hygro] Vector

    PDF (237 KB)

  • Certificate of Analysis

    Lookup Certificate of Analysis

    Storage Conditions

    -30C TO -10C

    Use Restrictions

    For Research Use Only. Not for Use in Diagnostic Procedures.

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