Journal
COMBINATORIAL CHEMISTRY & HIGH THROUGHPUT SCREENING
Volume 9, Issue 6, Pages 481-487Publisher
BENTHAM SCIENCE PUBL LTD
DOI: 10.2174/138620706777698535
Keywords
biotin depiction; fluorescence based assay; proteomics; protease assay
Funding
- NIAID NIH HHS [R44 AI056869-02] Funding Source: Medline
Ask authors/readers for more resources
We have developed a high throughput assay for the measurement of protease activity in solution. This technology will accelerate research in functional proteomics and enable biologists to streamline protease substrate evaluation and optimization. The peptide sequences that serve as protease substrates in this assay are labeled on the carboxy terminus with a biotin moiety and a fluorescent tag is attached to the amino terminus. Protease cleavage causes the biotin containing fragment to be detached from the labeled peptide fragment. Following the protease treatment, all biotin containing species (uncleaved substrates and the cleaved carboxy terminal fragment of the substrate) are removed by incubation with streptavidin beads. The cleaved fluorescently labeled amino terminal part of the substrate remains in solution. The measured fluorescence intensity of the solution is directly proportional to the activity of the protease. This assay was validated using trypsin, chymotrypsin, caspase-3, subtilisin-A, enterokinase and tobacco etch virus protease.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available