SpectraMax M3, M4, M5, and M5
e
Multi-Mode Microplate Readers
0112-0115 F 51
Optimizing Fluorescence Polarization Assays
Fluorescence polarization for SpectraMax M5 and M5
e
readers may only
be read from the top of a microplate. The plastic from a microplate will
affect 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 plates treated to minimize
binding, or polypropylene plates and/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 background's parallel and perpendicular readings
should be subtracted from the raw parallel and perpendicular readings
of each sample well before the mP calculation is performed. See the
SoftMax
®
Pro Software User Guide for set-up of background subtraction
in fluorescence polarization.
For best precision in assays using a low amount of fluorophore (for
example, <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 box, and
experiment with fewer flashes per well until acceptable precision and
speed are achieved.
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