HitHunter™ Protease Assays
There are about 475 known putative proteases in the human genome. Proteases participate in a wide variety of physiological processes by controlling complex proteolytic cascades. Many disease states which manifest as altered protease expression and substrate proteolysis are targets for therapeutic intervention. Protease inhibitor drugs, like ACE inhibitors and HIV inhibitors, act by inhibiting key proteases. Current efforts to design and screen for therapeutic protease inhibitors are being accelerated by the availability of structural information and several assay formats to screen, especially in the area of anticoagulation, neurodegenerative conditions, osteoporosis, type II diabetes and rheumatoid arthritis.
DiscoveRx offers two protease assays, but the format is conducive to custom EFC assays by inserting the protease cleavage sequence in a cyclized ED peptide.
Features and Benefits
» Non-antibody based in vitro assay for protease activity
• Amplified signal allows the detection of low activity proteases
» Minimal background
• Cyclized ED construct inhibits EFC signal
» Minimize Interference
• Luminescent detection reduces interference from fluorescent compounds, lowering false positives
Measuring protease activity with EFC technology
In the HitHunter™ Protease Assays, enzymes that recognize the Protease cleavage sequence will cleave the ED cyclic conjugate. If the conjugate is cleaved by a protease, it is free to recombine with EA, forming an active enzyme, which hydrolyzes the luminescent substrate to produce an easily detectable signal
The cyclic peptide that incorporates ED and the protease recognition sequence is unable to complement EA and no signal is generated, which minimizes background. As the protease cleaves the cyclic peptide, the linear form is able to complement EA and yields active ß-galactosidase to produce a detectable signal.
» Cyclic ED with protease cleavage sequence, EFC label
» EFC detection reagents
» Luminometer, Multimode Microplate Reader or CCD Camera