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
Related BioAssays
Related BioAssays
Targets
BioActive Compounds: 54
Depositor Specified Assays
| AID | Name | Type | Comment |
|---|---|---|---|
| 504435 | Broad Institute Small-molecule targeting of CFTR: PDZ Trafficking Interactions Inhibitor Probe Project | summary |
Description:
Keywords: CFTR, CAL, PDZ domain, fluorescence polarization
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.
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.
Protocol
In the Fluorescence Polarization (FP) assay, 5 uL of 5 uM CerCALP (Tagged version of the CAL PDZ protein) and 5 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 5.5 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 shaken, centrifuged, and 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 (Aurora 1536 well High Base Black Plate 00019180)
Dilute CerCALP in FP buffer to 5 uM. Dispense 5 uL/well using BioRaptr.
Dilute PRC34 in FP buffer to 5 uM. Dispense 5 uL/well using Combi NL.
Dispense 1 uL of PRC23 to positive control wells.
Dispense 1 uL of DMSO to neutral control wells.
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
1536 ARP Format (Aurora 1536 well High Base Black Plate 00019180)
Dilute CerCALP in FP buffer to 5 uM. Dispense 5 uL/well using BioRaptr.
Dilute PRC34 in FP buffer to 5 uM. Dispense 5 uL/well using Combi NL.
Dispense 1 uL of PRC23 to positive control wells.
Dispense 1 uL of DMSO to neutral control wells.
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
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
NORMALIZATION:
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.
PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the maximum of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 40.
PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 100.
PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T
Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T
Samples passing AT only were assigned an outcome of 2 (active) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T
tSamples passing PAR_T only were assigned an outcome of 2 (active) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
NORMALIZATION:
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.
PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the maximum of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 40.
PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 100.
PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T
Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T
Samples passing AT only were assigned an outcome of 2 (active) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T
tSamples passing PAR_T only were assigned an outcome of 2 (active) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
Result Definitions
| TID | Name | Description | Histogram | Type | Unit | |
|---|---|---|---|---|---|---|
| Outcome | The BioAssay activity outcome | Outcome | ||||
| Score | The BioAssay activity ranking score |  | Integer | |||
| 1 | REPRODUCIBILITY_COSINE_TRANSFORM | A measure of how well the activity reproduced across the two samples. Computed as the absolute value of the cosine between the 'replicate vector' (ScoreA, ScoreB ---as well as ScoreC and/or ScoreD where applicable) and the vector (1, 1) representing perfect reproducibility. NULL will appear in this column if a sample was not run in duplicate or if the data produced by one of the replicates was Invalid | | Float | ||
| 2 | REPLICATE_A_ACTIVITY_SCORE_20uM_(%) (20μM**) | The calculated activity for the indicated sample | | Float | % | |
| 3 | REPLICATE_B_ACTIVITY_SCORE_20uM_(%) (20μM**) | The calculated activity for the indicated sample | | Float | % |
** Test Concentration.
Additional Information
Grant Number: 1 R21 NS067613-01
Data Table (Concise)
Classification
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