Operating Procedures
0112-0127 B 111
Optimizing Fluorescence Polarization Assays
Fluorescence polarization (FP) can be read only from the top of a
microplate. The plastic from a microplate affects the light polarization,
precluding bottom reads and reading a covered plate.
Solid black plates are recommended for fluorescence polarization reads.
If the assay components seem to bind to the microplate, as evidenced by
poor mP dynamic range (small difference between bound and unbound
tracer), we suggest using polypropylene plates or adding a very small
amount of detergent, such as Tween-20, to the assay buffer. 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 parallel and
perpendicular readings of the background should be subtracted from the
raw parallel and perpendicular readings of each sample well before the
mP calculation is performed. For setup of background subtraction in
fluorescence polarization, see the SoftMax Pro Software User Guide.
For best precision in assays using a low amount of fluorophore (such as,
<5 nm fluorescein), set the PMT sensitivity to High and the number of
readings to 100. If faster read speed is required, be sure Settling Time is
Off in the SoftMax Pro Plate Settings dialog, and experiment with fewer
flashes per well until acceptable precision and speed are achieved.
FlexStation_manual.book Page 111 Tuesday, June 25, 2013 10:22 AM
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