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Protease-Glo™ Assay

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.
Visit the Protease-Glo™ Assay Design Tool to see how to generate your protease recognition site of interest in the pGloSensor™-10F Linear Vector and express the protein using cell-free translation. This web application makes proper oligo design fast and easy; simply enter your amino acid sequence of interest.

Specifications

What's in the box?

Item Part # SizeConcentrationAvailable Separately

2X Rapid Ligation Buffer

C671A 1 × 200μl View Product

Oligo Annealing Buffer

C838A 1 × 1ml

Bright-Glo™ Luciferase Assay Substrate

E263A 1 × 1 vial

Bright-Glo™ Luciferase Assay Buffer

E264A 1 × 10ml

pGloSensor™-10F Linear Vector

G946A 1 × 20μl View Product

pGloSensor™-10F(TEV) Control Plasmid

G947A 1 × 100μl

TnT® SP6 High-Yield Wheat Germ Master Mix

L326A 1 × 300μl View Product

T4 DNA Ligase

M180A 1 × 100u

DTT

P117B 1 × 250μl100mM View Product

Nuclease-Free Water

P119A 1 × 1,250μl View Product

ProTEV Buffer, 20X

V602A 1 × 1ml

ProTEV Plus

V610A 1 × 1,000u5u/μl View Product

SDS

Choose language:

Download SDSPDF (382 KB) – English (United States)

Use Restrictions

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

Storage Conditions

AA

See Protocol for storage recommendations.

Resources

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