Biology Reference
In-Depth Information
D
t
½
1
x
D
½
¼
(14.5)
a
2
r
ex
;
1
a
1
r
ex
;
2
½þ
D
t
A
t
ð
Þ
1
þ
R
TC
d
1
D
r
þ D
a
[
D
ref
]/[
A
ref
] must be obtained by a sep-
arate lux-FRET measurement from the so-called a tandem construct, where donor
and acceptor are in a known fixed stoichiometry. The ratios of excitation strengths
(donor/acceptor)
r
ex,
i
at excitation wavelengths
i
can be obtained from the spectral
parameters of the reference measurements by
D
a
¼
a
2
a
1
and
r
ex,2
r
ex,1
.
R
TC
¼
Here
D
r
¼
D
ref
A
ref
F
i;
re
D
ðÞ
e
i
D
e
A
ðÞ
F
i;
ref
A
Q
A
Q
D
r
ex
;i
e
i
A
¼
e
D
ðÞ
(14.6)
½
ðÞ
where e
D,
i
are the extinction coefficient,
e
D,A
(l) are the characteristic emission spec-
tra, and
Q
D,A
the fluorescence quantum yield of donor and acceptor.
Ef
D
is calculated by Eq.
(14.3)
from experimental quantities, without any additional
information. To calculate
Ef
A
and
x
D
by Eqs.
(14.4) and (14.5)
, further information from
a tandem construct with known donor-acceptor stoichiometry is required. Fluorescence
quantum yield of donor and acceptor is also necessary for lux-FRET calculations.
From lux-FRET, we receive the apparent FRET efficiencies
Ef
D
and
Ef
A
rather
than the true quantum efficiency of the energy transfer
E
. High
E
values however
help to improve the signal-to-noise ratio of the analysis. The fractions of DA com-
plexes scaled by total donor and acceptor concentration
f
D
and
f
A
are of potential
interest to study protein-protein interaction. By changing the donor mole fraction
x
D
, which can be assessed by varying the relative expression level of donor and
acceptor and keeping the total concentration constant, we get specific values for ap-
parent FRET efficiencies
Ef
D
and
Ef
A
. Amodel characterizing apparent FRET efficien-
cies
Ef
D
and
Ef
A
as a function of
x
D
for oligomeric structures has been defined:
;
x
D
x
D
x
n
1
D
x
n
1
D
Ef
D
¼
E
1
Ef
A
¼
E
1
(14.7)
1
Fitting this model to experimental data allows for the estimation of the true energy
transfer efficiency
E
and also provides information about the oligomerization state
n
(
Meyer et al., 2006
). When receptors interact and form dimers, the equations become
linear. For hetero-oligomerization, the model gets more complex (
Renner et al., 2012
).
14.2
MATERIALS / EXPERIMENTAL SETUPS
14.2.1
Reagents
Mouse N1E-115 neuroblastoma cells from America Type Culture Collection
(LGC Promochem)
Lipofectamine 2000 transfection reagent (Life Technologies, cat.no.11668-019)
Phenylmethylsulfonyl fluoride (PMSF) (Roth, cat.no.6367.1)
