Fluorescence Polarization with CAL-PDZ Measured in Biochemical System Using Plate Reader - 2109-02_Inhibitor_Dose_CherryPick_Activity_Set2
Assay Overview: A purified CAL PDZ domain construct is allowed to interact with a tetramethylrhodamine (TMR) labeled fluorescent reporter peptide that binds in the canonical peptide-binding groove. The disruption of the interaction is monitored using fluorescence polarization (FP). Fluorescence is measured at 535 nM in the S and P planes with the Perkin-Elmer Viewlux plate reader after 1 hour incubation at room temperature and the fluorescence polarization calculation (mP=1000(S-G*P)/(S+G*P) ) is determined. ..more
BioActive Compounds: 9
Keywords: CFTR, CAL, PDZ domain, fluorescence polarization, Cystic fibrosis
Assay Overview: A purified CAL PDZ domain construct is allowed to interact with a tetramethylrhodamine (TMR) labeled fluorescent reporter peptide that binds in the canonical peptide-binding groove. The disruption of the interaction is monitored using fluorescence polarization (FP). Fluorescence is measured at 535 nM in the S and P planes with the Perkin-Elmer Viewlux plate reader after 1 hour incubation at room temperature and the fluorescence polarization calculation (mP=1000(S-G*P)/(S+G*P) ) is determined.
Expected Outcome: Compounds that interfere with CAL-PDZ binding to the substrate will show a decrease in fluorescence polarization.
In the Fluorescence Polarization (FP) assay, 4 uL of 5 uM CALP (untagged version of the CAL PDZ protein) and 4 uL of 5 uM of T*-PRC34 (the reporter peptide) are dispensed into each well of a black Aurora 1536 assay ready plate (ARP) plate containing pre-dispensed compounds using a Beckman BioRaptr fluid dispensation apparatus. 1 uL of 4.5 mM unlabeled peptide is dispensed via BioRaptr to positive control wells. 1 uL of 11% DMSO is dispensed via the BioRaptr into neutral control wells. The plate is incubated for 1 hour at room temperature. The fluorescence in the S and P planes are read using the Viewlux plate reader (Perkin Elmer) and then the fluorescence polarization calculation is applied to determine mP value. Compounds are tested at a single concentration of 20 uM in the primary screen and at a range of doses for retests and subsequent studies.
1536 ARP Format (Aurora1536K High Base Black Plate 00019180)
Dilute CALP in FP buffer to 5 uM. Dispense 4 uL/well using BioRapTR.
Dilute PRC34 in FP buffer to 5 uM. Dispense 4 uL/well using Combi NL.
Dispense 1 uL of PRC23 to positive control wells using BioRapTR.
Dispense 1 uL of DMSO to neutral control wells using BioRapTR.
The assay plate is incubated at RT for 1h before reading.
Assay buffer: 25 mM NaH2PO4/ Na2HPO4*, 150 mM NaCl, 0.1 mM TCEP, pH 7.4., 0.1 mg/ml IgG, 0.5 mM Thesit
PRESENCE OF CONTROLS: Neutral control wells (NC; n=144) and positive control wells (PC; n=144) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
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: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v7.0.3) was applied to the normalized plate data.
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.
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)