eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 21
Figure 1: Illustration of FRET principle. Practically, one of the
binding partners in the interaction of interest is labeled with
a donor fluorophore such as a Europium chelate, whereas
the other partner is labeled with an acceptor fluorophore.
When direct labeling of the partners is not possible, the
molecule of interest is tagged or biotinylated and the pairing
is completed using streptavidin-coated donor or acceptor, or
anti-tag antibodies.
Classic fluorescence intensity uses short-lived
fluorophores such as fluorescein, with an emission
speed in the order of the nanoseconds. Excitation
and emission occur at specific wavelengths that
can be differentiated by a fluorescence reader.
However, excitation and emission happen at the
same time. If there is any amount of spectral overlap
between excitation and emission, as there usually
is, the reader will capture some of the excitation
fluorescence, resulting in background signal and
low signal-to-noise ratios.
or phosphorylation. The molecules under study
can be directly labeled with a donor or acceptor
fluorophore. Alternatively, a secondary reagent (for
example, an antibody) that binds to the molecule of
interest can be labeled with one of the fluorophores
for indirect detection. This provides great flexibility
on how to design an assay.
Time-Resolved Fluorescence (TRF)
FRET has a lower background signal than classic
fluorescence methods because the acceptor
emission is further apart from and does not have
a spectral overlap with the excitation pulse. The
donor and the acceptor, however, must have
good spectral overlap (the emission range of the
first must overlap with the excitation range of the
second), as well as good spectral resolution for a
specific signal to be measured. However, it is the
Time Resolved Fluorescence (TRF) technology
element that allows for the ultra-low background
advantage of TR-FRET.
TRF solves this by using long-lived inorganic
fluorophores as donors and adding a time delay
between excitation and measurement, which
means that the excitation signal is gone by the time
of the measurement, which decreases background
signals (Figure 2). TRF also uses excitation pulses
(not continuous excitation), so that a series of
measurements are repeated over time.
TRF
Figure 2: Illustration of TRF principle
21
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
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 1
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 2
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 3
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 4
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 5
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 6
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 7
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 8
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 9
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 10
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 11
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 12
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 13
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 14
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 15
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 16
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 17
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 18
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 19
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 20
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 21
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 22
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 23
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 24
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 25
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 26
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 27
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 28
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 29
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 30
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