The Protease-Glo Assay is a novel method to detect and measure protease activities using a genetically engineered firefly (Photinus pyralis) luciferase and represents one example of the GloSensor platform technology. 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. Visit the web application, Protease-Glo Assay Oligonucleotide Designer at: www.promega.com/techserv/tools/proteaseglo/ 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.|
Flexible: Use with P´ requiring proteases.Avoid Fluorescent Background Problems: Physical and chemical features of luminescence overcome problems due to fluorescence interference.Greater Sensitivity: Ease and dynamic range of luminescence.Open Platform System: Create your own recognition substrates.Interrogate Sequences: Excellent tool to determine optimal protease recognition sequences or effects of amino acid substitutions.Web Application: Makes proper oligo design fast and easy; simply enter your amino acid sequence of interest.See the Protease-Glo™ Assay Design Tool.