Molecular Devices SpectraMax Paradigm User Manual page 51

Blank Correction
Many fluorescence polarization assays use small fluorescent label concentrations in the
lower nm range. In this range, blank controls become significant when compared to samples.
A blank well contains everything used with the sample wells except the label and sample-
specific compounds. Do not use an empty well for a blank.
Background wells, containing all assay components minus the fluorophore, should be
tested. If the signal in the background wells is more than 1/10 the signal in the wells
containing fluorophore, then background wells should be run on each assay plate. The
average raw signal from the background's parallel and perpendicular readings must be
subtracted from the raw parallel and perpendicular readings of each sample well before the
mP calculation is done.
For optimum results, Molecular Devices recommends that you run replicates for all blanks,
controls, and samples. In this case, the blank value that can be subtracted is the average
value of all blanks.
Data Reduction
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.
Although the raw S and P values are the true actual values returned from the instrument, the
calculated Polarization (mP) and Anisotropy (r) values are treated as the raw data and
become the basis for further reduction calculations.
Polarization (mP) is calculated as follows:
mP = 1000 *
Anisotropy (r) is calculated as follows:
r =
The G factor, or grating factor, is used in fluorescence polarization to correct polarization
data for optical artifacts, converting relative mP data to theoretical mP data. Optical
systems, particularly with reflective components, pass light of different polarization with
different efficiency. G factor corrects this instrumental bias.
5014038 E
(parallel - (G * perpendicular))
(parallel + (G * perpendicular))
(parallel - (G * perpendicular))
(parallel + (2G * perpendicular))
Chapter 2: Read Modes and Read Types
51