Fluorescence Polarization Read Mode - Molecular Devices SpectraMax Paradigm User Manual

Z´ is the standard statistical parameter in the high-throughput screening community for
measuring the quality of a screening assay independent of test compounds. It is used as a
measure of the signal separation between the positive controls and the negative controls in
an assay.
The value of Z´ can be determined using the following formula:
Z´ = 1 –
where SD is the standard deviation, c+ is the positive control, and c– is the negative control.
A Z´ value greater than or equal to 0.4 is the generally acceptable minimum for an assay.
Higher values might be desired when results are more critical.
Z´ is not linear and can be made unrealistically small by outliers that skew the standard
deviations in either population. To improve the Z´ value, you can increase the quantity of
label in the sample, if acceptable for the assay, or increase the read time per well.

Fluorescence Polarization Read Mode

Fluorescence polarization (FP) mode measures the relative change of polarization of emitted
fluorescent compared to excitation light.
Fluorescence polarization detection is similar to fluorescence intensity, with the important
difference that it uses plane-polarized light, rather than non-polarized light. Plate readers
measure FP of the sample by detecting light emitted both parallel and perpendicular to the
plane of excitation.
By using a fluorescent dye to label a small molecule, its binding to a different molecule of
equal or greater size can be monitored through its speed of rotation.
When molecules are excited with polarized light, the polarization of the emitted light
depends on the size of the molecule to which the fluorophore is bound. Larger molecules
emit a higher percentage of polarized light, while smaller molecules emit a lower percentage
of polarized light because of their rapid molecular movement. For this reason FP is generally
used for molecular binding assays in high-throughput screening (HTS).
Fluorescence polarization mode returns two sets of data: one for fluorescence intensity
parallel (P) to the excitation plane, and the other for fluorescence intensity perpendicular (S)
to the excitation plane. These S and P values are used to calculate the Polarization (mP) and
Anisotropy (r) values in SoftMax Pro Software.
The Fluorescence Polarization data for a sample is evaluated based on its relative position
between the low and high control values. See
page 50.
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3(SD ) + 3(SD )
c+ c–
| Mean – Mean |
c+ c–
Chapter 2: Read Modes and Read Types
Analyzing Fluorescence Polarization Data on
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