Biology Reference
In-Depth Information
FIGURE 14.3
Flowchart showing steps to acquire various references according to different experimental
setups for quantitative lux-FRET measurement.
14.3.3.1
Setting individual protocols according to experimental setup
In the following, we describe experimental procedures to investigate the protein-
protein interaction using serotonin receptors 5-HT
1A
and 5-HT
7
tagged with mono-
meric versions of CFP and YFP as an example. However, the same strategy can be
applied to any protein of interest.
14.3.4
Spectroscopic measurement at a fluorescence
spectrometer (Fluorolog 3v.2.2.)
This system is highly suitable for FRET investigations in titration and time lapse ex-
periments for lysed cells and cell suspensions. Mouse N1E-115 neuroblastoma cells
were seeded in 35 mm dishes and cotransfected with plasmid DNAs encoding for
5-HT
1A
-CFP and 5-HT
7
-YFP receptors. 16 h after transfection, cells were washed
once with 1
PBS to remove residual medium and then resuspended in 2 ml buffer B
solution. All measurements were performed in 10 mm pathway quartz cuvettes. We
use front-face detection, where the fluorescence is collected from the sample's sur-
face. Typically, fluorescence signals are dwarfed by stray or scattered light from the
sample. We set chamber temperature to 25
C to compare results with microscope
experiments. During acquisition, the cell suspension was continuously stirred with
a magnetic stirrer. For reference measurements, cells were cotransfected with plas-
mid encoding a single fluorophore-tagged receptor (only donor and only acceptor).
