Biology Reference
In-Depth Information
Oligomerization
strong energy transfer between the FPs, which will result in a reduced molecular
brightness of the donor, in this case mTq2. This will lead to a more complicated con-
tribution of the CY dimer to the correlation curves. However, fluorescence lifetime
analysis showed a relatively small reduction of the fluorescence lifetime (
<
10%)
for the cotransfected (H
1
R-mTq2
H
1
R-sYFP2) cells with respect to H
1
R-mTq2-
transfected cells, and therefore, the FRET effect can be neglected.
In addition, the presence of endogenous, nonfluorescent H
1
R can affect the esti-
mated
K
D
values. Western blotting showed a relative low contribution of endogenous
H
1
R in resting HeLa cells in comparison to the average H
1
R-FP protein level after
successful transfection. However, when studies would be performed where cells are
stimulated by the histamine ligand, a significant increase of the endogenous H
1
R pro-
tein levels is expected (
Das et al., 2007
). In this situation, the fraction of dimers
where both monomers are fused to a FP will be lowered. This can be solved by using
siRNA strategies to lower the concentration of endogenous protein or the data can be
corrected as described by
Foo, Naredi-Rainer, Lamb, Ahmed, and Wohland (2012)
.
In this experiment, a single type of protein, H
1
R, is genetically fused to mTq2 or
sYFP2. Both plasmids, which are almost identical in code and size, are cotransfected
in HeLa cells. FCS experiments have shown that in this case, the production level of
both proteins is equal, validating Eq.
(11.9)
. However, when this equation has to be
adapted for experiments with two different kinds of proteins/constructs, a similar and
preferably known ratio of proteins is desired. We found that IRES and viral 2A pep-
tides can be used to coexpress proteins at a fixed ratio at the single-cell level
(
Goedhart et al., 2011
) that is very helpful for multicolor FCCS experiments as
described in this chapter.
þ
Acknowledgments
This work was supported by Middelgroot (834.09.003) and Echo (711.011.018) investment
grants and a VIDI fellowship awarded to M.P. (864.09.015) from the Netherlands Organisation
for Scientific Research (NWO). We thank Kevin Crosby and Kobus van Unen for sharing the
DNA constructs.
References
