A P P L I C AT I O N N O T E

Demonstrationof the capabilityof the EnVision
Nexus plate reader
in a fluorescencepolarization assay.

Introduction
The EnVision Nexus™ is a high-throughput multimode plate
reader purpose built for speed and sensitivity. As the next
generation of Revvity’s EnVision™ plate reader technology,
it continues to deliver the high sensitivity and optimal results
expected from EnVision instruments. Standard filter-based
technologies enable luminescence, absorbance, fluorescenceintensity, fluorescence polarization, and time-resolved
fluorescence (TRF) measurements. Optional add-on
technologies are also available for Ultrasensitive Luminescence,Alpha (amplified luminescent proximity homogeneous assay),and a dedicated TRF Laser, making the EnVision Nexus ahigh-performance instrument for all assay applications.
Fluorescence polarization technology measures changes in lightpolarization emitted by a fluorescent tracer in a sample and isAuthors
Adam Carlson
Revvity, Hopkinton MA

quite different from fluorescence intensity, which measures theintensity of emitted light at a specific wavelength. Fluorescencepolarization is a powerful tool used to study molecularinteractions by monitoring the change in rotational mobilityof fluorescently labeled molecules upon binding to a partnermolecule. This application note demonstrates the capability of
the EnVision Nexus to measure fluorescence polarization andcompares it to the EnVision 2105.

For research purposes only. Not for use in diagnostic procedures.

Demonstration of the capability of the EnVision Nexus plate reader in a fluorescence polarization assay.

Materials and methods

Fluorescence polarization assay information

Instrumentation

In a fluorescence polarization assay, the excitation of a

The EnVision Nexus multimode plate reader was evaluatedfor its ability to measure fluorescence polarization using
the Keap1:Nrf2 inhibitor screening assay kit (BPS Bioscience,#72020). The resulting fluorescence polarization values
were compared to values obtained from an EnVision 2105
multimode plate reader.

fluorophore bound to a small molecule with polarized lightleads to the emission of depolarized light due to the rapid
rotation rate of the small molecule complex. However, when
the fluorophore is bound to a larger molecule, the rotation
rate is slower, resulting in the emission of primarily polarizedlight. Fluorescence polarization is the measure of themolecular rotation that occurs in the time between excitationand emission. The measure of fluorescence polarization (mP)is calculated by the equation shown in Figure 2. Fluorescencepolarization assays require polarized excitation, one mirror
and two polarizing emission filters in the instruments,
typically labelled in the software as S (parallel or straight)
and P (perpendicular). The G-factor (g) is used to correct for
the effects of optical components like filters, polarizers,
and monochromators, which can affect polarization values.
The G-factor setting can be used to match the polarization toliterature values. This does not affect the change of measured
polarization over the assay, characterized by delta mP.
In the experiments shown here, the G-factor was set to 1 for
EnVision Nexus, where it is optional, but does not affectdata quality. On EnVision, the G-factor is set during again-optimization, which is important for good data quality.

Figure 2: mP calculation where I// is the intensity with polarizers
parallel to the excitation, I ⊥ is the intensity with polarizers
perpendicular to the excitation, and g is the g-factor.

Figure 1: Revvity’s multimode plate readers: EnVision Nexus
and EnVision 2105.

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2Demonstration of the capability of the EnVision Nexus plate reader in a fluorescence polarization assay.

The Keap1:Nrf2 Inhibitor Screening Assay Kit (BPSBioscience, #72020) is designed for the identificationof inhibitors of Keap1:Nrf2 binding using fluorescencepolarization. The manufacturer provided protocol wasfollowed using a final assay volume of 50 µL in a black,96-well ½-area OptiPlate (Revvity, #6002270). To determine
the effect of the inhibitor on the formation of Keap1:Nrf2complex, the Keap1 protein and the fluorescently labeled
Nrf2 peptide are incubated with or without the test inhibitorfor 30 minutes. The control small molecule inhibitor forthis assay was KI696 (Fisher Scientific, #50-202-8648)
which disrupts the interaction of Keap1 and Nrf2 and has
a reported IC50 of 0.093 µM in the Keap1:Nrf2 inhibitor
screening assay. Changes in the rotational mobility of thefluorescently labeled Nrf2 peptide are quantified using aplate reader capable of measuring fluorescence polarization.
When the labeled Nrf2 peptide is bound to Keap1 protein in
the absence of any inhibitor, the calculated mP value is high.
In the presence of an inhibitor, the Nrf2 peptide is no longer

Figure 3: Measurement height verification on the EnVision
Nexus using 1 nM fluorescein. Initial measurement height wasdetermined by the software after multiple reads of a single well.
Measurement heights above and below the initial optimal heightin 1 mm increments were set up to test on twelve replicate wells.
Standard deviation of the mP results at each manually tested
measurement height are shown.

bound to Keap1 and the calculated mP value is low.
Figure 4 shows the average mP values on the EnVision Nexus

Results and discussion
Fluorescence polarization results
Prior to running the Keap1:Nrf2 inhibitor screening assay,
the z height (measurement height) was optimized for both
instruments using 50 µL of a 1 nM fluorescein standard
prepared from a 100 nM stock solution (Revvity, #C557-100)
in a black, 96-well ½-area OptiPlate using the optimization

are higher than the EnVision 2105, however the calculated
IC50 values from the KI696 dose-response curves for each
instrument were similar: EnVision Nexus 0.077 µM andEnVision 2105 0.074 µM. The calculated IC50 values were
within an acceptable range of the manufacturer’s example
data (0.093 µM) for KI696 in the Keap1-Nrf2 inhibitor
screening assay. The overall shift to lower mP values on theEnVision 2105 is caused by the different G-factor settings,
this difference does not affect the information on the assay.

option available in the software for each plate reader.
After obtaining the optimized measurement height oneach instrument, one assay plate was set up for manualverification by loading twelve replicate wells with 50 µL
of 1 nM fluorescein per well. Measurement height settings
above and below the optimized height were evaluatedand the average mP value for fluorescence polarization
was calculated as described in the methods section.
The measurement height for each plate reader that yielded
the tightest standard deviation for the twelve replicates wasselected. In the case of the EnVision Nexus results seen in
Figure 3, the original optimized height of 9 mm was selected
as it performed the best.
Figure 4: Fluorescence polarization results from the Keap1:Nrf2
inhibitor screening assay. IC50 values of the resulting curves
were similar for both instruments: EnVision Nexus 0.077 µM andEnVision 2105 0.074 µM.

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3Demonstration of the capability of the EnVision Nexus plate reader in a fluorescence polarization assay.

In addition to calculating the mP values in the Keap1:Nrf2inhibitor screening assay, the delta mP value showing theConclusions

overall change in fluorescence polarization was plotted

The EnVision Nexus displayed superior fluorescenceto align the data more closely between instruments,

polarization performance which was achieved by utilizing

also eliminating differences due to G-factor settings.

the simple-to-follow default protocol and easy-to-install

Figure 5 shows similar curve fits for both instruments after

FITC fluorescence polarization filter module required by

calculation of the delta mP value where the negative control

the assay. After optimizing the measurement height for eachwells (no Keap1 protein) were subtracted from KI696 mP

instrument, the Keap1:Nrf2 inhibitor screening assay wasresults. The EnVision Nexus showed a larger delta mP assay

performed using the small molecule inhibitor KI696 as awindow, however the IC50 values remained similar:control. When comparing the mP results of the KI696

EnVision Nexus 0.077 µM and EnVision 2105 0.074 µM.

dose-response curve (Figure 4), the absolute mP signal ofthe EnVision Nexus was higher than the EnVision 2105,
however the curves were parallel resulting in similar
IC50 values on par with the manufacturer’s sample data.
Normalizing the fluorescence values to delta mP (Figure 5)
highlighted a benefit of the EnVision Nexus with a larger
assay window and tighter standard deviation. Despite thedifferences in curve shape in the delta mP graph, the IC50
results for KI696 remained similar for both instruments.
Overall, given the performance seen with the EnVision
Nexus and considerations such as the Kaleido™ softwareease-of-use and simple protocol set up, the EnVision Nexusdelivers the speed and accuracy needed for fluorescencepolarization assays.

Figure 5: Delta mP results from the Keap1:Nrf2 inhibitor
screening assay. The mP values for the negative control wells
were subtracted from all values and the resulting delta mPvalue was plotted. IC50 values were similar for both instruments:
EnVision Nexus 0.077 µM and EnVision 2105 0.074 µM.

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