eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 15

To summarize, the degree of polarization of a
fluorescent probe is a term that indicates to what
extent the light of excitation remains polarized.
When a small fluorophore is excited by polarized
light, its movement is fast, and it re-emits light
in all directions: it depolarizes the excitation
light and the degree of polarization is low. On
the contrary, reorientation of a large fluorophore
excited by polarized light is limited: the light
remains mostly polarized at re-emission and the
degree of polarization is high.
Experimentally, FP technology measures
fluorescence intensity emitted by the fluorophore
in the two planes of light that are parallel and
perpendicular relative to the plane of excitation.
The degree
of
fluorescence
polarization
(P)
is defined as the difference between the
fluorescence intensity parallel and perpendicular
relative to the plane of excitation, divided by the
total fluorescence intensity:
The G-factor is instrument-dependent and needs to
be determined by the investigator. The instrument
manual will contain information about how to
establish the G-factor.
FP Assays
II = Fluorescence Intensity parallel to plane of
excitation
I = Fluorescence Intensity perpendicular to plane
of excitation
Most instruments display fluorescence polarization
in units of mP in which 1 mP = 1000 P
FP assays measure changes in light polarization
that occur when a small fluorophore interacts with,
or dissociates from, a much larger partner. Any
small molecule which can be covalently labeled
with a fluorophore and can form a complex with a
larger partner is amenable to FP.
Several controls are necessary to conduct a
successful experiment:
Since (P) is a ratio of light intensities, it is a number
without dimension. Theoretical (P) values range
* " Blank " contains buffers and solvent but does
not contain the tracer or the binding partner.
It measures the small auto-fluorescence
generated by the reagents and should be
lower than the " Reference " . It is subtracted
from all measurements.
from -0.33 to 0.5 (-330 to 500 mP). Experimental
data typically range from 10 mP to around 300 mP.
Instruments achieve very precise measurements (P
± 0.002 or ± 2 mP).
The equation above assumes that light is
transmitted equally well through both parallel and
perpendicular channels. In practice, this is not true
and a correction must be made. The correction
factor is called the " G Factor " .
OR
15
T

https://bpsbioscience.com/assay-kits-faq#fp

eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development

Table of Contents for the Digital Edition of eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development