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
Target
| Sequence: Luciferase [Photinus pyralis] Description ..   Protein Family: Firefly luciferase of light emitting insects and 4-Coumarate-CoA Ligase (4CL) Related Protein 3D Structures |
Depositor Specified Assays
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| AID | Name | Type | Comment |
|---|---|---|---|
| 411 | qHTS Assay for Inhibitors of Firefly Luciferase | confirmatory | qHTS Assay for Inhibitors of Firefly Luciferase |
| 2228 | Thermal Shift Assay for Inhibitors of FLuc (Firefly Luciferase, Photinus pyralis) in Presence of ATP | screening | Thermal Shift Assay for Inhibitors of FLuc (Firefly Luciferase, Photinus pyralis) in Presence of ATP |
| 2229 | Thermal Shift Assay for Inhibitors of FLuc (Firefly Luciferase, Photinus pyralis) in Absence of ATP | screening | Thermal Shift Assay for Inhibitors of FLuc (Firefly Luciferase, Photinus pyralis) in Absence of ATP |
| 588342 | qHTS profiling assay for firefly luciferase inhibitor/activator using purifed enzyme and Km concentrations of substrates (counterscreen for miR-21 project) | confirmatory | qHTS Profiling Assay for Inhibitors of Firefly Luciferase with Km concentrations of substrate |
| 602357 | FLuc inhibitory activity for the follow-up compounds in a biochemical assay with Km concentrations of substrate | confirmatory | FLuc inhibitory activity for the follow-up compounds in a biochemical assay with Km concentrations of substrate |
| 602358 | FLuc inhibitory activity for the follow-up compounds in a biochemical assay with Km concentrations of substrate and 500microM CoASH | confirmatory | FLuc inhibitory activity for the follow-up compounds in a biochemical assay with Km concentrations of substrate and 500microM CoASH## |
| 602364 | FLuc inhibitory activity for the follow-up compounds in a biochemical assay with 1mM ATP | confirmatory | FLuc inhibitory activity for the follow-up compounds in a biochemical assay with 1mM ATP |
| 602365 | FLuc inhibitory activity for the follow-up compounds in a biochemical assay with 1mM D-luciferin | confirmatory | FLuc inhibitory activity for the follow-up compounds in a biochemical assay with 1mM D-luciferin |
| 602474 | FLuc inhibitory activity for the follow-up compounds in a biochemical assay with a commercial detection reagent - BriteliteTM Plus (PerkinElmer) | confirmatory | FLuc inhibitory activity for the follow-up compounds in a biochemical assay with a commercial detection reagent - BriteliteTM Plus (PerkinElmer)## |
| 602475 | FLuc inhibitory activity for the follow-up compounds in a biochemical assay with an in-house formulation of detection reagent | confirmatory | FLuc inhibitory activity for the follow-up compounds in a biochemical assay with an in-house formulation of detection reagent |
| 602476 | FLuc inhibitory activity for the follow-up compounds in a cell-based translational read-through assay | confirmatory | FLuc inhibitory activity for the follow-up compounds in a cell-based translational read-through assay |
| 602477 | FLuc inhibitory activity for the follow-up compounds in a cell-based assay to assess the activity of miR-21 | confirmatory | FLuc inhibitory activity for the follow-up compounds in a cell-based assay to assess the activity of miR-21 |
| 602478 | FLuc inhibitory activity for the follow-up compounds in a cell-based translational read-through assay (72 hour incubation) | confirmatory | FLuc inhibitory activity for the follow-up compounds in a cell-based translational read-through assay (72 hour incubation) |
| 624030 | Biochemical firefly luciferase enzyme assay for NPC | confirmatory | Biochemical firefly luciferase enzyme assay for NPC |
| 652016 | qHTS Assay for Inhibitors of Firefly Luciferase from the GSK Published Protein Kinase Inhibitor Set | confirmatory | 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).
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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