Biology Reference
In-Depth Information
where
N
C
and
N
Y
are the number of monomeric receptors labeled with mTq2 and
sYFP2, respectively.
N
CC
,
N
YY
, and
N
CY
correspond to the number of dimers with
a single or both types of FP labels.
For this experiment, we assume a double probability of observing a CY dimer
over a CC or YY dimer, and there is an equal production of mTq2- and sYFP2-
labeled receptors.
N
CY
¼
0
:
5
N
CC
¼
0
:
5
N
YY
(11.9)
Equations
(11.5)-(11.9)
are linked and fitted globally including a weight factor to
correct for the different detection volume sizes, as obtained from the calibration mea-
surements. In addition, the number of complexes is corrected for the fraction of over-
lap between the cyan and yellow detection volume as is estimated from the positive
control. From these numbers, the dissociation constant,
K
D
(
M¨der et al, 2007
), for
H
1
R dimerization is calculated. An example of our experimental values is presented
in
Table 11.1
. Due to the different expression levels among the cells, caused by the
transient transfection procedure, the interaction values are presented as percentage
complex in each detection channel and as apparent dissociation constant assuming
the absence of nonfluorescent and endogenous protein.
11.3
DISCUSSION
Quantitative measurements using FCCS require precise calibration measurements
because of the different detection volume sizes; the potential existence of nonmature
dark FPs and photobleaching can affect the results. Dimerization could induce a
Table 11.1
Line-FCCS Analysis of HeLa Cells Cotransfected with H
1
R-mTq2 and
H
1
R-sYFP2 and the Control Samples
Diffusion Time
t
x
(ms)
% Interaction
(N
CY
/N
Xtotal
)
Apparent K
D
(nM)
n
()
Construct(s)
H
1
R-mTq2
308
00
n.a.
3
H
1
R-sYFP2
3711
00
n.a.
3
H
1
R-mTq2 þ
329
124
64691
5
H
1
R-sYFP2
3912
185
mTq2-p63
2410
992
n.a
3
sYFP2
(fusion, pos.)
2813
981
CAAX-mTq2 þ
1.20.7
11
35123984
2
CAAX-sYFP2
(neg.)
1.10.7
00
The data are analyzed using Eqs.
(11.5)-(11.9)
and the obtained parameters are presented for the
detection channel corresponding to the FP in the construct. The errors presented are SEM. n.a.,
nonapplicable.
