Thursday, January 24, 2013

Applications Thursday: A Novel Fluorescent Intensity uHTS Assay that Utilizes Physiological Protein Substrates

A recent assay performed at Sanford-Burnham Center for Chemical Genomics entitled: 'Single concentration confirmation of uHTS inhibitor hits of Sentrin-Specific Protease 8 using Nedd8 Protein Substrate' describes a fluoresence intensity assay using BMG LABTECH's PHERAstar FS designed to find inhibitors of an enzyme involved in the neddylation pathway. Targeting the neddylation machinery is attractive due to the role of this machinery in indirect regulation of cell cycle progression, DNA replication and DNA damage response mechanisms.

DNA_damaged_by_carcinogenic_2-aminofluorene_AF_.jpg
The application is straightforward enough involving detection of deconjugated aminocoumarin from a Nedd8-AMC substrate. The released aminocoumarin is readily detected with ex/em at 350/450  using a microplate reader such as the PHERAstar FS from BMG LABTECH. Using the full length Nedd8 should reveal inhibitors that bind to the actual conformation of SENP8 when it is bound to substrate.


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