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BioAssay: AID 590

qHTS Assay for Spectroscopic Profiling in A350 Spectral Region

The rate of false hits is dramatically reduced by the qHTS approach (Inglese et al, PNAS, 103, 1147 (2006)) as spurious high- or low-response wells are quickly revealed when plotted in the context of concentration response. Occasionally, the artifactual effect may originate from the compounds own spectral or other biophysical properties which generally tend to track its assay concentration. more ..
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 Tested Compounds
 Tested Compounds
All(58464)
 
 
Active(6427)
 
 
Inactive(43468)
 
 
Inconclusive(8735)
 
 
 Tested Substances
 Tested Substances
All(59094)
 
 
Active(6462)
 
 
Inactive(43867)
 
 
Inconclusive(8765)
 
 
 Related BioAssays
 Related BioAssays
AID: 590
Data Source: NCGC (SPEC113)
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-02-12
Modify Date: 2007-03-01

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 6427
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Description:
NIH Molecular Libraries Screening Centers Network [MLSCN]
NIH Chemical Genomics Center [NCGC]

NCGC Assay Overview:
The rate of false hits is dramatically reduced by the qHTS approach (Inglese et al, PNAS, 103, 1147 (2006)) as spurious high- or low-response wells are quickly revealed when plotted in the context of concentration response. Occasionally, the artifactual effect may originate from the compounds own spectral or other biophysical properties which generally tend to track its assay concentration. Pre-screening/profiling the library with respect to the compounds spectral properties should help one exclude false positives early on. While the most informative fluorescent profiling data set is obtained by performing a full excitation and emission scan of each well by using a monochromator-based plate reader, such an approach is practical only on a very limited scale as scanning each well requires long time. An alternative approach, applied here, is to perform plate reader scans using selected excitation and emission wavelength pairs to generate a multidimensional relational database that correlates compound concentration and the respective relative fluorescence intensity at a given wavelength set. A standard curve of Alexa 350 (A 350) present on each plate as six-point concentration series, was used to compute a normalized fluorescence response, termed dye-equivalent concentration, for each compound at each concentration.
Protocol
NCGC Assay Protocol Summary:

Buffer:
100 mM Tris buffer, pH 8.0

Dye standard plate:
The wells in columns 1 through 4 of a Kalypsys polypropylene 1536-well compound plate were filled with standard solutions of fluorophore corresponding to the spectral region of interest. Solutions of A 350 were placed in the following wells: I1, J1: 3 mM, I2, J2: 300 uM, I3, J3: 30 uM, I4, J4: zero (DMSO), K1, L1: 3 uM, K2, L2: 900 nM, K3, L3: 300 nM, K4, L4: zero (DMSO).

Assay Steps:
Six uL of buffer were dispensed to 1536-well Greiner black plates. Compounds and dye standards (23 nL) were transferred via Kalypsys PinTool. The plates were incubated for 15 min at room temperature, and then read on ViewLux (Perkin-Elmer) High-throughput CCD imager using 340 nm and 450 nm excitation and emission filter set.

Keywords: NIH Roadmap, MLSCN, MLI, MLSMR, qHTS, NCGC, A350, A-350, Fluorescent Profiling.
Comment
Compound Classification and Ranking:

1. A log of maximum dye equivalent concentration was determined for each sample. Samples that were -9 or below in log of max dye equivalency were declared as inactives. Samples between -8 and -9 were inconclusive and samples greater than -8 were declared as active. An active is considered to be a sample that showed significant fluoresence at this particular dye's excitation and emission wavelengths.

2. For all inactive samples, PUBCHEM_ACTIVITY_SCORE is 0. For all active samples, ranking of dye equivalency was normalized from 100uM (score of 100) to 1nM (score of 0). A higher score is given to samples that exhibited more fluoresence in this assay.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Log of MaxDyeEquivalencyLog of the maximum dye equivalent concentration observed for sampleFloat
2MaxDyeEquivalencyThe maximum dye equivalent concentration observed for sampleFloat
3DyeEquivalency at 0.5nMDye equivalent concentration at given cocentrationFloat
4DyeEquivalency at 1.1nMDye equivalent concentration at given cocentrationFloat
5DyeEquivalency at 2.5nMDye equivalent concentration at given cocentrationFloat
6DyeEquivalency at 5.5nMDye equivalent concentration at given cocentrationFloat
7DyeEquivalency at 12.3nMDye equivalent concentration at given cocentrationFloat
8DyeEquivalency at 27.5nMDye equivalent concentration at given cocentrationFloat
9DyeEquivalency at 0.06uMDye equivalent concentration at given cocentrationFloat
10DyeEquivalency at 0.14uMDye equivalent concentration at given cocentrationFloat
11DyeEquivalency at 0.31uMDye equivalent concentration at given cocentrationFloat
12DyeEquivalency at 0.69uMDye equivalent concentration at given cocentrationFloat
13DyeEquivalency at 1.54uMDye equivalent concentration at given cocentrationFloat
14DyeEquivalency at 3.44uMDye equivalent concentration at given cocentrationFloat
15DyeEquivalency at 7.69uMDye equivalent concentration at given cocentrationFloat
16DyeEquivalency at 17.2uMDye equivalent concentration at given cocentrationFloat
17DyeEquivalency at 38.46uMDye equivalent concentration at given cocentrationFloat
18Compound TypeNCGC designation for compound stage: 'qHTS ECL (Exploratory)', 'NIHSMR (MLSCN)', 'Compound Followup', 'Compound Verification', 'Probe Optimization'String

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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