Activator for delta FosB/delta FosB homodimer Measured in Biochemical System Using Plate Reader - 2072-01_Activator_SinglePoint_HTS_Activity
Keywords: transcriptional factor, delta FosB homodimer, AP-1 DNA binding, activator screening, biochemical fluorescence polarization assay ..more
BioActive Compounds: 3330
Keywords: transcriptional factor, delta FosB homodimer, AP-1 DNA binding, activator screening, biochemical fluorescence polarization assay
Assay Overview: The assay uses fluorescence polarization to measure the binding of purified recombinant delta FosB to a TAMRA-labeled (fluorescent) oligonucleotide derived from the AP-1 consensus site of the Cdk5 gene (TAMRA-CDK5), a confirmed target gene. The assay relies on the tumbling of the fluorescence labeled Cdk5 oligo in solution that slows down when the much larger delta FosB protein binds to it, which causes an increase in the fluorescence polarization signal. Any compound that increases fluorescence polarization signal of a solution with low concentration of delta FosB and TAMRA-CDK5 is considered as active compound. These compounds can work directly by increasing the affinity of delta FosB for AP-1 DNA, or indirectly by increasing the affinity of delta FosB for itself thereby stabilizing dimers which in turn increases the affinity of delta FosB for AP-1 DNA.
Mouse delta FosB is cloned into expression vector pET28a(+) and transformed into E.coli Rosetta2(DE3) strain
Delta FosB is extracted from inclusion body and purified through Ni-NTA affinity purification. His tag is removed by TEV protease cleavage.
Purified protein is stored in the buffer of 20 mM Tris-HCl (pH8.0), 0.1 M NaCl, 1mM DTT, 10% Glycerol, 0.5 mM PMSF at -80 degrees C.
1. Prepare 2x low-dose delta FosB and 2x high-dose delta FosB in FP buffer
2. Dispense 10uL/well of 2x high-dose delta FosB and of FP buffer to 384-well ARPs by BioRAPTR (Beckman) based on plate map
ARP is prepared by dispense 50nL/well of 10mM compound to 384-well assay plate (Perkin Elmer, Cat#: 6008269)
3. Dispense 10uL/well of 2x low-dose delta FosB to ARPs by multi-drop combi (Thermo Scientific)
4. Incubate delta FosB with compound at room temperature for 10mins
5. Dispense 10uL/well of 100nM TAMRA-CDK5 in FP buffer to the plate by multi-drop combi (Thermo Scientific)
6. Incubate at 25 degrees C for 15mins
7. Read the plate on EnVision (PerkinElmer) with 20 time flashes. The excitation wavelength is 540nm and emission wavelength is 590nm with the dichroic mirror of D555/D595.
Final concentration: 1xlow-dose delta FosB: 30nM, 1xhigh-dose delta FosB: 600nM, TAMRA-CDK5: 50nM
FP buffer: 20mM HEPES pH7.5, 50mM NaCl, 1mM DTT (DTT added freshly)
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.
EXPECTED OUTCOME: Active compounds result in increasing readout signal.
The raw signals of the plate wells were normalized using the 'Stimulators Minus Neutral Controls' 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.
This was set as equal to the mean of the normalized 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 16.
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.
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 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T
Samples passing PAR_T only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
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Data Table (Concise)