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Protease-Glo™ Assay

Detect and Measure Protease Activities

  • Luminescent assay
  • Quickly create your own protease substrates
  • Easy quantitation, high sensitivity and wide dynamic range

Size

Catalog number selected: G9451

$ 1,135.00 Your price: Log In

Protease-Glo™ Assay
1 each
$ 1,135.00
Your price: Log In

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.

Protocols

Specifications

What's in the box?

Item Part # SizeConcentrationAvailable Separately

2X Rapid Ligation Buffer

C671A 1 x 200μl View Product

Oligo Annealing Buffer

C838A 1 x 1ml

Bright-Glo™ Luciferase Assay Substrate

E263A 1 x 1 vial

Bright-Glo™ Luciferase Assay Buffer

E264A 1 x 10ml

pGloSensor™-10F Linear Vector

G946A 1 x 20μl View Product

pGloSensor™-10F(TEV) Control Plasmid

G947A 1 x 100μl

TnT® SP6 High-Yield Wheat Germ Master Mix

L326A 1 x 300μl View Product

T4 DNA Ligase

M180A 1 x 100u

DTT

P117B 1 x 250μl100mM View Product

Nuclease-Free Water

P119A 1 x 1,250μl View Product

ProTEV Buffer, 20X

V602A 1 x 1ml

ProTEV Plus

V610A 1 x 1,000u5u/μl View Product

SDS

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Certificate of Analysis

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Use Restrictions

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

Storage Conditions

AA

See Protocol for storage recommendations.

Patents and Disclaimers

BY USE OF THIS PRODUCT, RESEARCHER AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE STATEMENT. If the researcher is not willing to accept the conditions of this limited use statement, and the product is unused, Promega will accept return of the unused product and provide the researcher with a full refund.
NOT FOR USE IN LIVE CELLS OR LIVE ANIMALS—For use in live cells or animals contact Promega regarding the availability of licenses. Researchers may use this product and its derivatives in their own research solely for the extracellular (in vitro) determination of protease activity. Researchers shall have no right to modify or otherwise create variations of the nucleotide sequence of the circularly permuted luciferase reporter of the product and derivatives except that researchers may add oligonucleotides at the site corresponding to the free ends of the linearized product. In addition, researchers must either: (1) use reagents purchased from Promega for all (a) determinations of luminescence activity, (b) extracellular transcription of DNA sequences, and (c) extracellular translation of transcripts; or (2) contact Promega to obtain a license for the use of the product and its derivatives in conjunction with luminescent assay reagents not purchased from Promega. Researchers may transfer derivatives to others provided that recipients agree to be bound by the conditions of this limited use statement prior to such transfer. No other use or transfer of this product or derivatives is authorized without the prior express, written consent of Promega. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE WITH REGARDS TO THE PRODUCT. The terms of this agreement shall be governed under the laws of the State of Wisconsin, USA.

U.S. Pat. No. 8,008,006 and European Pat. No. 1341808.

Licensed under U.S. Pat. No. 5,910,628.

Resources

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