ARNT-TACC3: counter AlphaScreen Measured in Biochemical System Using Plate Reader - 2158-02_Inhibitor_Dose_CherryPick_Activity
Keywords: Aryl hydrocarbon receptor nuclear translocator (ARNT), coil coil coactivators (CCC), basic helix-loop-helix/per-ARNT-Sim (bHLH/PAS), hypoxia, xenobiotic exposure ..more
BioActive Compounds: 617
Keywords: Aryl hydrocarbon receptor nuclear translocator (ARNT), coil coil coactivators (CCC), basic helix-loop-helix/per-ARNT-Sim (bHLH/PAS), hypoxia, xenobiotic exposure
Assay Overview: A dual tagged protein Alphascreen detection (Perkin Elmer) is used for this secondary assay. This in vitro assay relies on luminescence proximity between "donor" and "acceptor" beads, requiring that two beads localize within 200 nm for the singlet O2-mediated process to occur in high efficiency. This assay is looking for artifacts of Alphascreen signal. Instead of two individually tagged proteins being added to the mixture, a dual tagged His-ARNT PASB-GST protein is added along with compound. Using affinity tags to recruit beads into close proximity, any compound-mediated decreases in luminescence are monitored to indicate candidates that may be artificially decreasing the signal. In this assay, compounds that are decreasing luminescence are considered artifacts and will be eliminated as probe candidates. The positive control is the His-ARNT protein alone.
Expected Outcome: It is expected that most compounds will not interfere with Alphascreen signal (no change in signal). Compounds that are decreasing luminescence are considered artifacts and will be eliminated as probe candidates.
1.Dual tagged His6-ARNT-GST protein (In Alpha storage buffer, 273 microM)
2.Ni2+ acceptor beads, 10 mg/ml (PerkinElmer, Nickel Chelate AlphaLISA(R) Acceptor Beads 10 mg, Product number: AL108M)
3.GST donor beads, 10 mg/ml (PerkinElmer, AlphaScreen(R) Glutathione Donor Beads, Product number: Product number: 6765302 for 25mg pack)
4.Alpha storage buffer: 50 mM Tris, pH 7.5; 100 mM NaCl; 10% glycerol; 1 mM DTT, 0.02 % Tween 20
5.Alpha screening buffer: 50 mM Tris, pH 7.5; 100 mM NaCl; 1% glycerol; 0.02 % Tween 20, 1 mM DTT (freshly added); 0.1% BSA (freshly added)
6.Assay Plates: Perkin Elmer 384 well AlphaPlate (cat. no 6005350)
1.Add 15 microL dual tagged His6-ARNT-GST protein (0.668 microM) to compound wells and neutral control wells of 384 plate using Combi (final concentration 0.668 microM))
2.Add 7.5 microL of Alpha screening buffer to positive control wells using Combi
3.Incubate for 1 hr. at room temperature
4.Add 10 uL/well of Alphabead mixture (25 microg/mL) using Combi (final concentration 10 microg/mL)
5.Incubate for 1 hr. at room temperature in the dark
6.Read plate using on Envision plate reader using protocol: ARNT Alphascreen 384
PRESENCE OF CONTROLS: Neutral control wells (NC; n=36) and positive control wells (PC; n=36) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
The compounds were assayed in multiple independent instances using an identical protocol; each instance is called a 'test'. For each test, the following analysis was applied:
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.
Once the data for each test was processed, the test number was appended to all column headers in that test's data set. The individual test results were then aggregated as follows:
1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:
a. '2' (active) when all test outcomes were '2' (active), or
b. '1' (inactive) when all test outcomes were '1' (inactive), or
c. '3' (inconclusive) when the test outcomes were mixed.
2. The final ACTIVE_CONCENTRATION (AC) was set as follows:
a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the mean of the constituent test active concentrations;
b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1 or 3, AC was left empty.
3. The final PUBCHEM_ACTIVITY_SCORE was calculated based on the aggregated ACTIVE_CONCENTRATION, using the same logic described above for individual test scores.
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)