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pNL3.2.NF-kB-RE[NlucP/NF-kB-RE/Hygro] Vector

by Promega
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  • The pNL3.2.NF-?B-RE[NlucP/NF-?B-RE/Hygro] Vector contains five copies of an NF-?B response element (NF-?B-RE) that drives transcription of a destabilized form of NanoLuc® luciferase, an engineered small (23.3kDa) luciferase fusion protein. The NlucP reporter consists of NanoLuc® luciferase with a C-terminal fusion to PEST, a protein destabilization domain, which responds more quickly and with greater magnitude to changes in transcriptional activity than unmodified NanoLuc® luciferase.

    NanoLuc® (Nluc) luciferase is a small enzyme (19.1kDa) engineered for optimal performance as a luminescent reporter. The enzyme is about 100-fold brighter than either firefly (Photinus pyralis) or Renilla reniformis luciferase using a novel substrate, furimazine, to produce high intensity, glow-type luminescence. The luminescent reaction is ATP-independent and designed to suppress background luminescence for maximal assay sensitivity.

    Applications

    • Transcription regulation.
    • Virus-cell interactions.
    • Compound screening.
    • GPCR signaling.
    • Cell signaling.

    About the NanoLuc® Luciferase Reporter Enzyme

    NanoLuc® luciferase in the form of NanoLuc®-PEST (NlucP) closely couples protein expression to changes in transcriptional activity and increased signal-to background ratios. The PEST sequence appended to Nluc destabilizes the reporter protein, make NanoLuc® luciferase more responsive to changes in the cell. Luminescence is linearly proportional to the amount of NanoLuc® protein over a 1,000,000-fold concentration range, with a signal half-life ?2 hours when detected with Nano-Glo® Luciferase Assay Reagent.

    NanoLuc® luciferase possesses a number of physical properties that make it an excellent reporter protein:

    • very small, monomeric enzyme (171 amino acids; 513bp)
    • high thermal stability (Tm = 60°C)
    • active over a broad pH range (pH 6–8)
    • no post-translational modifications or disulfide bonds
    • uniform distribution in cells
    • emission spectrum well suited for bioluminescence resonance energy transfer (BRET; ?max = 465nM).

    NanoLuc® luciferase is made available in a variety of plasmids designed for use in reporter gene assays of transcriptional control and with each of the NanoLuc® forms (unfused Nluc, PEST destabilized NlucP, and secreted secNluc). The different pNL variations are designed for the following:

    • pNL1: cloning of a known or putative promoter region
    • pNL2: cloning of a known or putative promoter region and establishment of a stable cell line through Hygromycin selection
    • pNL3: cloning of a binding site or response element not in need of a basic promoter (such as are present in the pNL3.2.NF-?B-RE vector)
    • Control plasmids for the unfused, PEST-destabilized and secreted Nluc forms also are available.

    The pNL vector series uses a pGL4-based backbone for easy sequence transfer from existing plasmids. This backbone design also reduces anomalous results by removing many transcription factor binding sites and other potential regulatory elements. The NlucP gene is codon optimized and has had many potential regulatory elements or other undesirable features removed (such as common restriction enzyme sites).

    Sequence Information

    pNL3.2.NF-?B-RE[NlucP/NF-?B-RE/Hygro] Vector GenBank® Accession Number JQ513377 and vector sequence text file.

  • Protocols

    Complete Protocol

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    pNL3.2.NF-?B-RE[NlucP/NF-?B-RE/Hygro] Vector Product Information

    PDF (173 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.

  • Resources

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