Molecular Devices SpectraMax Paradigm User Manual page 50

SpectraMax Paradigm Multi-Mode Detection Platform User Guide
Applications of Fluorescence Polarization
Fluorescence polarization measurements provide information on molecular orientation and
mobility, and are generally used to quantify the success of a binding reaction between a
smaller labeled ligand and a binding site at a much larger or immobilized molecule. FP can
also be used to quantify the dissociation or cleavage of the labeled ligand from a binding site.
FP is a homogeneous microplate assay technique and requires only mixing and measuring—
no wash steps are required as in an ELISA. It can also be miniaturized, which makes it useful
for high-throughput screening applications.
You can use the Protocol Manager in the SoftMax Pro Software to quickly find and open a
predefined protocol.
More protocols and updated protocols can be downloaded from the Knowledge Base on the
Molecular Devices support web site (www.moleculardevices.com/support) or from the
protocol sharing web site (www.softmaxpro.org).
The following detection cartridges have fluorescence polarization read mode capability:
Fluorescence Polarization (FP) Detection Cartridges, see page 125
Analyzing Fluorescence Polarization Data
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.
FP assays in microplates are generally designed with two control samples:
LOW control sample: minimal polarization value resulting from unbound labeled ligand
only
HIGH control sample: maximum polarization value resulting from bound labeled ligand
only
The FP data for a sample is evaluated based on its relative position between the low and high
control values. Total intensity can also be determined from the raw data and is proportional
to the quantity of label in a sample.
Analyzing and interpreting fluorescence polarization data generally consists of the following:
Blank Correction on page 51
Data Reduction on page 51
Data Qualification and Validation on page 52
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