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Protease-Glo Assay

by Promega
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  • Protease-Glo™ Assay

    A novel method to detect and measure protease activities using a genetically engineered firefly (Photinus pyralis) luciferase, representing one example of the GloSensor™ platform technology. The Protease-Glo™ Assay can be used to:

    • Measure specificity of proteases or protease recognition sequences.
    • Compare related proteases against the same substrate (e.g., viral strains).
    • Determine protease substrate specificity (length of essential sequence, what specific amino acids are essential).
    • Screen for protease inhibitors and determine inhibitor potencies (IC50).

    The assay uses a circularly permuted firefly luciferase, the GloSensor™-10F protein, with a protease recognition site as the protease substrate. This assay system allows rapid generation of protease substrates through molecular cloning and coupled transcription/translation cell-free expression, thus enabling the facile evaluation of protease function. Oligonucleotides encoding a protease recognition sequence are designed and cloned into the GloSensor™-10F gene located on a linearized vector. The GloSensor™ protein containing the protease site of interest is then synthesized in a cell-free protein expression system and subsequently used as a protease substrate. Cleavage of the protease recognition sequence leads to activation of the GloSensor™ protein and light emission. The level of luminescence correlates to protease activity. The Protease-Glo™ Assay has the advantage of a bioluminescent readout, which provides easy quantitation, high sensitivity and wide dynamic range. The physical and chemical features of luminescence overcome problems due to fluorescence interference.

     

    References

    1. Fan, F. et al. (2008) Novel genetically encoded biosensors using firefly luciferase. ACS Chem. Biol. 3, 346–51.
    2. Wigdal, S. et al. (2008) A novel bioluminescent protease assay using engineered firefly luciferase. Curr. Chem. Genomics 1, 94–106.
    3. Fan, F. and Wood, K.V. (2007) Bioluminescent assays for high-throughput screening. Assay Drug. Dev. Technol. 5, 127–136.
  • Protocols

    Complete Protocol

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    Protease-Glo™ Assay Technical Manual

    PDF (640 KB)

  • Certificate of Analysis

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    Storage Conditions

    LESS THAN -65C

    Use Restrictions

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

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