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BioAssay: AID 2309

Probe Summary for Inhibitors and Stabilizers of Firefly Luciferase

Firefly (Photinus pyralis) luciferase is one of the most commonly used transcriptional reporters. It is considered one of the more dynamically responsive reporters - that is, responsive to changes in reporter transcription - as it requires no post-translational modifications and is enzymatically active directly after protein synthesis (Wood, 1998). With no endogenous expression in mammalian cells and without the requirement of external excitation, it offers very little background signal, thus generally providing excellent signal to background for most assays. ..more
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AID: 2309
Data Source: NCGC (FLUC200)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-01-28
Modify Date: 2013-02-07
Target
Related Experiments
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AIDNameTypeComment
411qHTS Assay for Inhibitors of Firefly LuciferaseConfirmatorydepositor-specified cross reference: qHTS Assay for Inhibitors of Firefly Luciferase
2228Thermal Shift Assay for Inhibitors of FLuc (Firefly Luciferase, Photinus pyralis) in Presence of ATPScreeningdepositor-specified cross reference: Thermal Shift Assay for Inhibitors of FLuc (Firefly Luciferase, Photinus pyralis) in Presence of ATP
2229Thermal Shift Assay for Inhibitors of FLuc (Firefly Luciferase, Photinus pyralis) in Absence of ATPScreeningdepositor-specified cross reference: Thermal Shift Assay for Inhibitors of FLuc (Firefly Luciferase, Photinus pyralis) in Absence of ATP
588342qHTS profiling assay for firefly luciferase inhibitor/activator using purifed enzyme and Km concentrations of substrates (counterscreen for miR-21 project)Confirmatorydepositor-specified cross reference: qHTS Profiling Assay for Inhibitors of Firefly Luciferase with Km concentrations of substrate
602357FLuc inhibitory activity for the follow-up compounds in a biochemical assay with Km concentrations of substrateConfirmatorydepositor-specified cross reference: FLuc inhibitory activity for the follow-up compounds in a biochemical assay with Km concentrations o
602358FLuc inhibitory activity for the follow-up compounds in a biochemical assay with Km concentrations of substrate and 500microM CoASHConfirmatorydepositor-specified cross reference: FLuc inhibitory activity for the follow-up compounds in a biochemical assay with Km concentrations o
602364FLuc inhibitory activity for the follow-up compounds in a biochemical assay with 1mM ATPConfirmatorydepositor-specified cross reference: FLuc inhibitory activity for the follow-up compounds in a biochemical assay with 1mM ATP
602365FLuc inhibitory activity for the follow-up compounds in a biochemical assay with 1mM D-luciferinConfirmatorydepositor-specified cross reference: FLuc inhibitory activity for the follow-up compounds in a biochemical assay with 1mM D-luciferin
602474FLuc inhibitory activity for the follow-up compounds in a biochemical assay with a commercial detection reagent - BriteliteTM Plus (PerkinElmer)Confirmatorydepositor-specified cross reference: FLuc inhibitory activity for the follow-up compounds in a biochemical assay with a commercial detect
602475FLuc inhibitory activity for the follow-up compounds in a biochemical assay with an in-house formulation of detection reagentConfirmatorydepositor-specified cross reference: FLuc inhibitory activity for the follow-up compounds in a biochemical assay with an in-house formula
602476FLuc inhibitory activity for the follow-up compounds in a cell-based translational read-through assayConfirmatorydepositor-specified cross reference: FLuc inhibitory activity for the follow-up compounds in a cell-based translational read-through assa
602477FLuc inhibitory activity for the follow-up compounds in a cell-based assay to assess the activity of miR-21Confirmatorydepositor-specified cross reference: FLuc inhibitory activity for the follow-up compounds in a cell-based assay to assess the activity of
602478FLuc inhibitory activity for the follow-up compounds in a cell-based translational read-through assay (72 hour incubation)Confirmatorydepositor-specified cross reference: FLuc inhibitory activity for the follow-up compounds in a cell-based translational read-through assa
624030Biochemical firefly luciferase enzyme assay for NPCConfirmatorydepositor-specified cross reference: Biochemical firefly luciferase enzyme assay for NPC
652016qHTS Assay for Inhibitors of Firefly Luciferase from the GSK Published Protein Kinase Inhibitor SetConfirmatorydepositor-specified cross reference: qHTS Assay for Inhibitors of Firefly Luciferase from the GSK Published Protein Kinase Inhibitor Set
Description:
NCGC Assay Overview:

Firefly (Photinus pyralis) luciferase is one of the most commonly used transcriptional reporters. It is considered one of the more dynamically responsive reporters - that is, responsive to changes in reporter transcription - as it requires no post-translational modifications and is enzymatically active directly after protein synthesis (Wood, 1998). With no endogenous expression in mammalian cells and without the requirement of external excitation, it offers very little background signal, thus generally providing excellent signal to background for most assays.

Previous profiling work to determine the prevalence of compounds that affect FLuc enzymatic activity (PubChem AID 411) identified that ~3% of the MLSMR library (then at ~72K) inhibited FLuc (Auld et al., 2008). More recent profiling efforts using KM concentrations of FLuc substrates indicate that the number of compounds that inhibit FLuc is even greater than this estimate. In cell-based FLuc reporter gene assays, it has been found that compounds that inhibit FLuc can cause either inhibition or activation of FLuc (Thorne et al., 2010). Whether inhibition or activation of FLuc is demonstrated in a cell-based assay by a FLuc inhibitory compound depends on the potency of the compound, the mode of inhibition of the compound, the assay format and protocol, and the detection reagent used. FLuc inhibitory compounds that cause activation in a cell-based FLuc reporter gene assay have been found to interact with the FLuc protein in the cell, thereby stabilizing it, increasing its protein half-life, and allowing it to accumulate intracellularly (Thompson et al., 1991). An example of such compounds are a subset of 3,5-diaryl oxiadiazoles that are potent FLuc inhibitors that can increase the melting temperature of FLuc (Auld et al., 2010).
Protocol
Please see AIDs for individual BioAssay protocol details: AID 411, 2228, 2229, 588342, 602357, 602358, 602364, 602365, 602474, 602475, 602476, 602477, 602478.
Comment
This summary assay will be updated as additional data is made available in PubChem.
Additional Information
Grant Number: U54

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