Biology Reference
In-Depth Information
fluorimeter, for example, Varian Cary Eclipse. Note that it is possible to study
more dilute samples by increasing the slit widths of the fluorimeter with the
associated decrease in specificity. We tend to avoid samples where it is
necessary to increase the slit widths further than 5 nm for excitation and emission.
If this is the case, then more concentrated samples should be produced, for example,
by ultracentrifugation followed by resuspension in a smaller volume. Low-volume
cuvettes, for example, 50
m
l, are ideal for these assays. Experiments should be
performed in triplicate.
Once data have been collected, a number of corrections are performed (see
Fig. 18.2
). Firstly, the appropriate liposome background spectrum (1 or 2) is
subtracted from each NTS1 containing sample of the corresponding excitation wave-
length to generate spectra 3-7. This background may require scaling if the
liposome fluorescence spectra have significantly different intensities to the spectra
to be corrected although this should not normally be the case. Any difference
likely arises due to the detergent that is present in the protein-containing samples,
but not the controls, during reconstitution resulting in differently sized liposomes.
It is important to scale using a region unaffected by the donor or acceptor emissions.
This subtraction eliminates any effects of scattering or endogenous lipid fluores-
cence to create a “zero” background. Next, bleedthrough and crosstalk must be cor-
rected for. In the following example, NTS1-CFP is the donor and NTS1-YFP the
acceptor.
Bleedthrough is the emission of the donor (CFP) at the wavelength at which the
acceptor (YFP) emits. This is subtracted by the following:
1.
Scale the NTS1-CFP spectrum (3) so that the fluorescence at the maximum
(typically
475 nm) is equal to the fluorescence at the same wavelength
in the NTS1-CFP
NTS1-YFP spectrum (6) to generate spectrum 8.
2.
Subtract spectrum 8 from spectrum 6. This removes bleedthrough and the
remaining signal is a combination of FRET and crosstalk (9).
þ
Crosstalk is the direct excitation of the acceptor (YFP) with the wavelength used to
excite the donor (CFP; 440 nm). This is subtracted by the following:
1.
Scale the NTS1-YFP spectrum (5) so that the fluorescence at the maximum
(
NTS1-YFP spectrum (7). This
compares the amount of NTS1-YFP in the two samples. Determine the scaling
factor required to do this (spectrum 5-spectrum 7).
2.
Multiply spectrum 4 (NTS1-YFP) by this scaling factor to determine the
extent of crosstalk in the NTS1-CFP
525 nm) is the same as in the NTS1-CFP
þ
þ
NTS1-YFP sample; this generates
spectrum 10.
3.
Subtract spectrum 10 (crosstalk) from spectrum 9 (FRET
þ
crosstalk) to
determine the specific FRET from this experiment (11).
Once these corrections have been made, the remaining signal is FRET. It is advisable
to conduct all experiments in triplicate and to use the average of the apparent FRET
efficiency values (see succeeding text).
