fluorescence change over the course of the experiment. Furthermore, buffers should not be stored in plastic containers as
leaching of fluorescent softeners can occur.
2. Fluorescence is detected at 90
from the incident light source.
3. The excitation and emission slit widths should be set so as to
not overexpose your sample and the photomultiplier tubes
(PMTs) to light, respectively. Proteins and ligands can become
photobleached over the course of an experiment, reducing the
amount of emitted light over time, thus it is advised that the
amount of incident light is decreased to a level that provides a
measurable output (see Note 6). Additionally, the larger the
excitation slit widths are, the larger the wavelength range
around the excitation wavelength is that is allowed to pass
through and excite the sample. Thus, depending on the range
of excitation wavelengths, there may be other fluorophores
excited that could contribute to the observed fluorescence
output. An example of this is the excitation of tryptophan and
tyrosine in proteins, both of which can be excited at 280 nm,
but tryptophan primarily excited at 295 nm. This can be beneficial as it allows selective excitation of tryptophan residue
(opposed to also exciting the more abundant tyrosine) in the
protein of interest (if the protein only has one tryptophan, is a
variant with only one tryptophan, or only has one tryptophan
within the proximity of the ligand binding site) and the measurement of the distance between a specific donor tryptophan
residue and acceptor fluorophore based on the FRET efficiency.
This is a specific example, but the logic can be applied to other
FRET pair systems to measure distances on a nanoscale.
4. The excitation wavelength is dependent on the donor fluorophore used in the experiment. Here we used FRET between
tryptophan/tyrosine residues in our protein of interest (HflX)
excited at 280 nm and measure the fluorescence emission of the
Mant group covalently attached to the bound nucleotide.
Other donor fluorophores can be used and the excitation
wavelength adjusted accordingly. Additionally, the emission
scan should not overlap the excitation wavelength (e.g., exciting at 420 nm and measuring emission from 350 to 500 nm)
and emission is generally measured starting 10–15 nm from the
excitation wavelength (also see Note 3 for slit width considerations). This is to prevent overexposing the PMT, causing damage to the instrument.
5. The protein concentration used in the experiment is dependent
on the intensity of the fluorescence signal but will determine
the lowest possible K d that can be measured. As a general rule,
this lower boundary is equal to the concentration of protein
used in the experiment as this ensures that the measured
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Harland E. Brandon and Hans-Joachim Wieden
leaching of fluorescent softeners can occur.
2. Fluorescence is detected at 90
from the incident light source.
3. The excitation and emission slit widths should be set so as to
not overexpose your sample and the photomultiplier tubes
(PMTs) to light, respectively. Proteins and ligands can become
photobleached over the course of an experiment, reducing the
amount of emitted light over time, thus it is advised that the
amount of incident light is decreased to a level that provides a
measurable output (see Note 6). Additionally, the larger the
excitation slit widths are, the larger the wavelength range
around the excitation wavelength is that is allowed to pass
through and excite the sample. Thus, depending on the range
of excitation wavelengths, there may be other fluorophores
excited that could contribute to the observed fluorescence
output. An example of this is the excitation of tryptophan and
tyrosine in proteins, both of which can be excited at 280 nm,
but tryptophan primarily excited at 295 nm. This can be beneficial as it allows selective excitation of tryptophan residue
(opposed to also exciting the more abundant tyrosine) in the
protein of interest (if the protein only has one tryptophan, is a
variant with only one tryptophan, or only has one tryptophan
within the proximity of the ligand binding site) and the measurement of the distance between a specific donor tryptophan
residue and acceptor fluorophore based on the FRET efficiency.
This is a specific example, but the logic can be applied to other
FRET pair systems to measure distances on a nanoscale.
4. The excitation wavelength is dependent on the donor fluorophore used in the experiment. Here we used FRET between
tryptophan/tyrosine residues in our protein of interest (HflX)
excited at 280 nm and measure the fluorescence emission of the
Mant group covalently attached to the bound nucleotide.
Other donor fluorophores can be used and the excitation
wavelength adjusted accordingly. Additionally, the emission
scan should not overlap the excitation wavelength (e.g., exciting at 420 nm and measuring emission from 350 to 500 nm)
and emission is generally measured starting 10–15 nm from the
excitation wavelength (also see Note 3 for slit width considerations). This is to prevent overexposing the PMT, causing damage to the instrument.
5. The protein concentration used in the experiment is dependent
on the intensity of the fluorescence signal but will determine
the lowest possible K d that can be measured. As a general rule,
this lower boundary is equal to the concentration of protein
used in the experiment as this ensures that the measured
282
Harland E. Brandon and Hans-Joachim Wieden