Bookmark and Share
BioAssay: AID 1776

Profiling compound fluorescence on GSH Beads with 488 nm excitation and 530 nm emission

Innate fluorescence from testing compounds could potentially affect response measurements during screening. For the multiplex screen of BCL-2 (AID:950,951, 952, 1007, 1008, 1009) glutathione labeled beads were coated with GST Bcl-fusion proteins, onto which fluorescent Bim binds. In addition, non-protein coated glutatione beads were also present in each assayed well. The fluorescence of these more ..
_
   
 Tested Compounds
 Tested Compounds
All(194829)
 
 
Active(489)
 
 
Inactive(194183)
 
 
Unspecified(158)
 
 
 Tested Substances
 Tested Substances
All(194920)
 
 
Active(489)
 
 
Inactive(194273)
 
 
Unspecified(158)
 
 
 Related BioAssays
 Related BioAssays
AID: 1776
Data Source: NMMLSC (UNM_BCL_GSHBead_COMPOUND_FLUORESCENCE)
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-05-20
Modify Date: 2011-03-28

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 489
Related Experiments
Show more
AIDNameTypeProbeComment
950Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-2.Screening depositor-specified cross reference: HTS Multiplex screen of Bcl-2 on GSH beads
951Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-B.Screening depositor-specified cross reference: HTS Multiplex screen of Bcl-B on GSH beads
952Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-W.Screening depositor-specified cross reference: HTS Multiplex screen of Bcl-W on GSH beads
1007Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-XL.Screening depositor-specified cross reference: HTS Multiplex screen of Bcl-XL on GSH beads
1008Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bfl-1Screening depositor-specified cross reference: HTS Multiplex screen of Bfl-1 on GSH beads
1009Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Mcl-1Screening depositor-specified cross reference: HTS Multiplex screen of Mcl-1 on GSH beads
1693Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions via Bim (BCL2-like 11)Summary1 depositor-specified cross reference: Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interaction
432HTS discovery of chemical inhibitors of anti-apoptotic protein Bfl-1Confirmatory same project related to Summary assay
621TR-FRET secondary assay for HTS discovery of chemical inhibitors of anti-apoptotic protein Bfl-1Confirmatory same project related to Summary assay
748High Throughput Fluorescence Polarization Screen for Bcl-B Phenotype ConvertersConfirmatory same project related to Summary assay
1320Multiplexed dose response screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bfl-1.Confirmatory same project related to Summary assay
1322Multiplexed dose response screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-XL.Confirmatory same project related to Summary assay
1324Profiling Assay to determine GST-GSH interactions in multiplex bead-based assays (HPSMTB buffer)Confirmatory same project related to Summary assay
1327Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-B protein.Confirmatory same project related to Summary assay
1328Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-2Confirmatory same project related to Summary assay
1329Multiplexed dose response screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Mcl-1.Confirmatory same project related to Summary assay
1330Multiplexed dose response screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-W.Confirmatory same project related to Summary assay
2075Confirmation dose response of hits from multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-2Confirmatory same project related to Summary assay
2077Confirmation dose response of hits from multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-BConfirmatory same project related to Summary assay
2080Confirmation dose response of hits from multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bfl-1Confirmatory same project related to Summary assay
2081Confirmation dose response of hits from multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-WConfirmatory same project related to Summary assay
2084Confirmation dose response of hits from multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-XLConfirmatory same project related to Summary assay
2086Confirmation dose response of hits from multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Mcl-1Confirmatory same project related to Summary assay
504598Dose response of powder sourced compounds for small molecule regulators of Bcl-2 family protein interactions, panel upload with wildtype and mutant Bfl1 and wildtype BclB.Other same project related to Summary assay
504627Bcl-2 family members Fluorescence polarization assay with Set1 of powder compoundsOther same project related to Summary assay
588575SAR analysis of selective Bcl-B inhibitors using fluorescence polarization assayConfirmatory same project related to Summary assay
588578SAR analysis of selective Bcl-B inhibitors using a Fluorescence Polarization Bcl-XL/Bim-BH3 AssayConfirmatory same project related to Summary assay
588716Isothermal titration calorimetry (ITC) with Bcl-B and compounds active in primary screenConfirmatory same project related to Summary assay
720677SAR analysis of selective Bcl-B inhibitors using fluorescence polarization assay, set 2Confirmatory same project related to Summary assay
Description:
University of New Mexico Assay Overview:
Assay Support: NIH 1X01 MH079850-01
HTS to identify small molecule regulators of Bcl-2 family protein interactions
PI: Larry Sklar, Ph.D.
Assay Implementatiion: Peter Simons Ph.D, Susan Young MS, Anna Waller Ph.D, Mark Carter M.S.
Target Team Leader for the Center: Larry Sklar, Ph.D., (lsklar@salud.unm.edu)

Assay Background and Significance:

Innate fluorescence from testing compounds could potentially affect response measurements during screening. For the multiplex screen of BCL-2 (AID:950,951, 952, 1007, 1008, 1009) glutathione labeled beads were coated with GST Bcl-fusion proteins, onto which fluorescent Bim binds. In addition, non-protein coated glutatione beads were also present in each assayed well. The fluorescence of these glutathione beads in the presence of testing compounds or just DMSO were measured with excitation at 488 nm and emission at 530 +/- 20 nm. For the flow cytometer to measure the fluorescence, the compound would be non-specifically bound externally to the bead. The fluorescence measurements were calibrated with the aid of calibration beads, which are assessed to have certain molecules of equivalent soluble fluorophore (MESF) per bead.
Protocol
The multiplex assay consists of seven bead sets of glutatione labeled bead (ThermoFisher Scientific product numbers XPR-1687-XPR-1696); six of the bead sets have been pre-incubated with GST Bcl-fusion protein target and one unlabeled bead set. These bead sets are labeled with different intensities with red fluorescence, hence are distinguishable from one another. After overnight incubation with GST-tagged proteins, the beads are washed in HPSMTB buffer (30mM HEPES, 100mM KCl, 20mM NaCl, 1mM MgCl(2), 0.01% Tween-20, 0.1% BSA). The bead sets are then resuspended in HPSMTB and stored on ice until the 384-well plates are assembled using the Biomek FXp, Automated work station;(5 microliters of bead mixture, 0.1 microliters of test compound, and 5 microliters of 100 nM F-Bim in HPSMTB). The final test compound concentration is 10 microM. The control wells contain DMSO instead of test compound.
Sample analysis is conducted with the HyperCyt(R) high throughput flow cytometry platform. The HyperCyt(R) system interfaces a flow cytometer and autosampler for high-throughput microliter-volume sampling from 384-well microtiter plates [Kuckuck, et al. 2001]. Flow cytometric data of light scatter and fluorescence emission at 530 +/- 20 nanom (FL1) and 665 +/- 10 nm (FL8) are collected on a Cyan Flow Cytometer (Dako). Time-resolved data is acquired as a single data file that is subsequently analyzed using IDLeQuery software (software developed inhouse by Dr. Bruce Edwards) that merges flow cytometry data files with compound worklist files generated by HyperSip software. The raw data are parsed in IDLeQuery to produce annotated fluorescence summary data for each well. Gating based on forward scatter (FS) and side scatter (SS) parameters is used to identify singlet bead populations. Additional gating based on FL8 (red) emission distinguishes the beads coated with different proteins. Per bead population, the median channel fluorescence (MCF) from FL1 (green) channel is calculated.
Calculations:
Autofluorescence of test compounds were calculated on the basis of the median fluorescence intensity (MFI) detected in the green fluorescence channel (530 +/- 20 nM) and the subtraction of the autofluorescence of the uncoated avidin bead, as shown in the following equation:
Fluorescence = MFI_CMPD - MFI_DMSO
where MFI_CMPD is the MFI of avidin beads in the presence of test compound and MFI_DMSO is the MFI of beads in the presence of DMSO (control well).
The Fluorescence was calibrated to kMESF (kiloMESF, 1000*MESF) utilizing Bangs Labs calibration beads by the following equation:
kMESF_bead = Slope*Fluorescence + Y-intercept
where Slope and Y-intercept are from linear fit of MFI measurements of the 5 different calibration beads with known MESF.
The calculated kMESF for each compound is the reported Activity Score, with the exception of when the kMESF was greater than 100 the Score was set at 100. A compound was noted active if the fluorescence was greater than the average plus three standard deviations of the entire screen. For this assay that kMESF cutoff was 86 (25.5+3*20). Therefore any compound with Activity Score > 86 is active.
Keywords:
NIH Roadmap, NMMLSC, flow cytometry, compound fluorescence, Bcl-2 family
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1COMPOUND_KMESF (10μM**)Compound innate fluorescence on glutathione beads in kilo Molecules as Equivalent Soluble FluorophoresFloat

** Test Concentration.
Additional Information
Grant Number: NIH 1X01 MH079850-01

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