Tecan Infinite™ F500 , Fluorescence Polarization

Application Note
Screening of Vitamin D Receptor ligands with PolarScreen Red
(Invitrogen)
Tecan Infinite F500 , Fluorescence Polarization
Introduction
The Vitamin D Receptor (VDR)
The vitamin D receptor (VDR) is a member of the nuclear
receptor (NR) gene superfamily. NRs mediate cellular
responses to a broad range of small molecular weight, nonpeptide
signals, including endogenous hormones and
metabolites as well as xenobiotic compounds (1).
The VDR is a ligand-dependent transcription factor and it
mediates the biological action of vitamin D in humans. It plays
a crucial role in maintaining the structural integrity of bones
through its effects on calcium and phosphorous homeostasis.
Furthermore it modulates the immune response, cell
proliferation and cell differentiation. Research on the VDR is
of very high importance for the treatment of bone disorders
and psoriasis, as well as for the treatment of immune
disorders and malignant tumors (2).
Assay Principle
Invitrogen’s PolarScreen Vitamin D Receptor Competitor
Assay, Red, is a fluorescence polarization (FP)-based
competitor assay that provides a sensitive and robust method
for high-throughput screening of potential vitamin D receptor
ligands. The kit uses the novel, tight-binding fluorescent VDR
ligand Fluoromone VDR Red and full-length human VDR
(untagged) in a homogenous mix-and-read assay format (2).
VDR is added to a fluorescent VDR ligand (Fluoromone
VDR Red) to form a receptor/tracer complex resulting in a
high polarization value. This complex is then added to
individual test compounds in microwell plates. Competitors
will displace the tracer from the complex, causing the
fluorescent ligand to tumble more rapidly during its
fluorescence lifetime and resulting in a low polarization value
(see figure 1).
Bound Tracer:
High
Figure 1: General scheme of PolarScreen NR Assays
Free Tracer:
Low
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Application Note
Material and methods
Instrument
Tecan Infinite TM F500 filter-based multimode micro plate
detection system
Microplates
384 Flat bottom, black Polystyrol microwell plate (Corning)
Reagents
PolarScreen TM Vitamin D Receptor Competition Assay, Red
(Invitrogen, PV4569)
Assay Procedure
[1] Prepare VDR Red Screening Buffer as described in the
protocol.
[2] Prepare a solution of calcitrol at 100x of the final desired
maximum starting concentration using DMSO (for these
experiments, a stock of 100 µM calcitrol was prepared for
starting concentration in the assay of 1 µM).
[3] For the “maximum agonist” controls, dilute the 100x
agonist solution from step 3 to 2x using VDR Red
screening Buffer.
[4] Prepare a 16-point 100x dilution series of agonist by
serially diluting the 100x agonist solution from step 3
three-fold using DMSO. Repeat for 15 dilutions plus the
starting stock.
[5] Dilute each 100x agonist serial dilution from step 5 to 2x
using VDR Red Screening Buffer. Mix by pipetting up
and down.
[6] Transfer 20 µl of each of the 2x agonist serial dilutions to
duplicate rows in a 384-well assay plate.
[7] Prepare 2x VDR/Fluoromone VDR Red Complex by
adding first fluoromone and then VDR to a glass
container on ice, to a final concentration of 2 nM
Fluoromone and 1.4 nM VDR. Use 20 µl per well in
appropriate wells.
[8] Plate should contain, when fully prepared, calcitrol
dilution curve, wells containing VDR plus fluoromone
alone (no calcitrol), and G-Factor Reference (Fluoromone
tracer alone in buffer, no VDR) and G-Factor Blank
(buffer alone) control wells.
[9] Cover the plate and incubate at room temperature.
Plates were incubated for 2 hours.
Measurements
Plate
Measurement
[COS384fb.pdf]
Mode Fluorescence Polarization
Excitation Wavelength 535 [25] nm
Emission Wavelength 590 [20] nm
Gain 86 Calculated From: C24
Number oF Reads 10
Integration Time 40 µs
Lag Time 0 µs
Settle Time 0 ms
Mirror (Automatic) Dichroic 560 (e.g. Cy3)
Table 1: Infinite TM F500 measurement parameters for PolarScreen TM
Vitamin D Receptor Competition Assay
Results and discussion
The measurements were performed with the Infinite TM F500
and the Tecan Ultra 384 for comparison (Table 2). Figure 2
shows the titration curve for determining the EC50 of
Calcitriol.
Figure 2. PolarScreen VDR Red on Infinite TM F500. Measured
polarization values were plotted against Calcitriol dilution series.
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Application Note
Ultra 384 Infinite F500
LOW mP 238 245
HIGH mP 382 388
Delta mP 144 144
EC 50 3.1 2.8
Table 2: PolarScreen VDR Red – comparison of results obtained with
Tecan Ultra 384 and Infinite TM F500.
Figure 2 and Table 2 clearly demonstrate an excellent
performance of Infinite TM F500 in regard to the test
requirements for the PolarScreen Far-Red Kinase Assay.
Tecan Infinite TM F500 shows a well comparable performance
with Tecan Ultra 384 that was used for reference
measurements.
Conclusion
This technical note describes the successful performance of
Invitrogen´s PolarScreen Vitamin D Receptor Competitor
Assay, Red on the Tecan Infinite TM F500 filter based
multimode detection system. In regard to the obtained data
the instrument is perfectly performing according to the given
assay requirements.
Acknowledgements
We express our acknowledgements to Dr. Mark Koeppel
and Dr. Kurt Vogel from Invitrogen/Madison, who
provided us with excellent support during the validation
of the Infinite TM F500.
Literature
[1] Invitrogen homepage: http://www.invitrogen.com
[2] PolarScreen Vitamin D Receptor Competitor Assay,
Red, Kit insert (Cat.No.PV4569)
List of abbreviations
VDR vitamin D receptor
NR nuclear receptor
FP Fluorescence Polarization
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