Biology Reference
In-Depth Information
Oligomerization
centrifugation and resuspended in 50 mM PBS. Cells are lysed by passage
through a French pressure cell. Soluble protein is obtained after centrifugation at
20,000 g for 30 min at 4
C. The protein binds to the chitin beads and after
extensive wash with PBS, the FPs are eluted from the column by incubating
the beads overnight in 50 mM dithiothreitol. Protein purity is checked on
SDS-PAGE.
4.
Solution of purified N1 plasmid DNA encoding for the protein fusions
mTurquoise2-p63-sYFP2, human H
1
R-mTurquoise2 or H
1
R-sYFP2.
5.
DMEM growth medium or phenol-free DMEM growth medium (Gibco)
supplemented with GlutaMAX (Gibco).
6.
Live cell incubator at 37
C with 5% CO
2
.
7.
Lipofectamine and Opti-MEM transfection agents (Invitrogen).
8.
Sterile circular (ø 24 mm, size 1) coverslips (Menzel-Gl¨ser).
9.
Attofluor coverslip holder (Invitrogen).
10.
Six-well container (Greiner).
11.
96-well microtiter plates with borosilicate bottom (Whatman).
12.
Confocal laser scanning microscope FV1000 (Olympus) equipped with lasers
and MPD detectors as described in detail in the succeeding text.
13.
FFS data processor 2.3 software package (Scientific Software Technologies
Center).
14.
Ptu converter 0.40 (van Leeuwenhoek Centre for Advanced Microscopy).
15.
Matlab 7.0 (MathWorks).
11.1.2
Fluorescent proteins
Because FCS is a method that relies on the analysis of relative fluorescence fluctu-
ations, the average concentration of fluorescent molecules should be kept low, typ-
ically between 1 nM and 1
M. While the molecules pass through the detection
volume, as many photons as possible should be detected. As shown by
Koppel
(1974)
, the molecular brightness of the fluorophore is a key parameter to obtain
high-quality FCS data. Since our work focuses on studying cellular signaling within
living cells, genetically encoded FPs are the most suitable dyes. The two FPs selected
on the basis of highest molecular brightness, minimal absorbance spectrum overlap, fast
chromophore maturation, and good photostability are mTurquoise2 (mTq2) (
Goedhart
et al., 2012
)andsYFP2(
Kremers, Goedhart, van Munster, & Gadella, 2006
).
m
11.1.3
Microscope
Data are acquired using an Olympus IX81 inverted microscope with a Fluoview1000
scan and confocal detection head, controlled by FV3.1 software (Olympus). The
514 nm line of a continuous wave Ar
þ
laser (Omnichrome) is combined with a puls-
ing laser diode of 440 nm (PicoQuant) operated at a rate of 20 MHz, as controlled by
a Sepia II laser driver unit (PicoQuant). The light intensity is attenuated 10 times by a
neutral density filter and guided via a D440/514 primary dichroic mirror (Chroma)
