fraction-bound will be at 50%. See Note 6 for rationale behind
the intensity of the fluorescence signal.
6. A measurable output is such that the baseline fluctuation in
signal is not equal to or greater than the increase/decrease in
fluorescent signal from the experiment. This is valid for both
equilibrium and pre-steady state experiments.
7. The concentrations of nucleotide or ligand in general to add
over the course of the titration should be based on an approximate K d if known, otherwise should span several orders of
magnitude. Additions do not need to be of the same volume
at each point as long as the volume added is noted (see Note 8).
The number of additions (data points) one should perform for
each titration curve is dependent on the quality of the data such
that a defined curve can be fit with Eq. (6) (see Subheading
3.1.2, step 15). Typically, 15–20 ligand concentrations that are
distributed equally above and below the anticipated K d will
provide an accurate initial measurement of the K d .
8. A dilution of the protein occurs with each addition of ligand
which will result in a decrease in fluorescent signal as a function
of protein concentration. When analyzing the data this must be
taken into account. This is done by calculating the adjusted
concentration of the protein after each addition and normalizing to protein concentration (see Eq. 10). The measured
fluorescence intensity is multiplied by the correction factor
(CF). V I is the initial volume of the reaction and V D is the
diluted volume. The CF will need to be calculated for each
dilution performed and measured.
CF ¼ V D =V I
ð10Þ
9. The background fluorescence should be subtracted from each
subsequent measurement to correct for any trace fluorescence
from the buffer alone. This is typically carried out after correcting for the dilution change with each addition of ligand (see
Note 8).
10. The fluorescence maximum depends on the physical properties
of the protein being studied. Therefore, the wavelength chosen
to be plotted should be consistently the highest from several
trials.
11. Many steps in this protocol are specific to the KinTek SF-2004
stopped-flow apparatus and appropriate changes should be
considered when using other stopped-flow devices. Consult
the user manual or manufacturing company for specific operational procedures to the apparatus.
12. Long-pass wavelength filters allow only light of wavelengths
greater than the indicated wavelength to pass through. Light of
Fluorescence-Based Equilibrium and Pre-Steady State Methods
283
the intensity of the fluorescence signal.
6. A measurable output is such that the baseline fluctuation in
signal is not equal to or greater than the increase/decrease in
fluorescent signal from the experiment. This is valid for both
equilibrium and pre-steady state experiments.
7. The concentrations of nucleotide or ligand in general to add
over the course of the titration should be based on an approximate K d if known, otherwise should span several orders of
magnitude. Additions do not need to be of the same volume
at each point as long as the volume added is noted (see Note 8).
The number of additions (data points) one should perform for
each titration curve is dependent on the quality of the data such
that a defined curve can be fit with Eq. (6) (see Subheading
3.1.2, step 15). Typically, 15–20 ligand concentrations that are
distributed equally above and below the anticipated K d will
provide an accurate initial measurement of the K d .
8. A dilution of the protein occurs with each addition of ligand
which will result in a decrease in fluorescent signal as a function
of protein concentration. When analyzing the data this must be
taken into account. This is done by calculating the adjusted
concentration of the protein after each addition and normalizing to protein concentration (see Eq. 10). The measured
fluorescence intensity is multiplied by the correction factor
(CF). V I is the initial volume of the reaction and V D is the
diluted volume. The CF will need to be calculated for each
dilution performed and measured.
CF ¼ V D =V I
ð10Þ
9. The background fluorescence should be subtracted from each
subsequent measurement to correct for any trace fluorescence
from the buffer alone. This is typically carried out after correcting for the dilution change with each addition of ligand (see
Note 8).
10. The fluorescence maximum depends on the physical properties
of the protein being studied. Therefore, the wavelength chosen
to be plotted should be consistently the highest from several
trials.
11. Many steps in this protocol are specific to the KinTek SF-2004
stopped-flow apparatus and appropriate changes should be
considered when using other stopped-flow devices. Consult
the user manual or manufacturing company for specific operational procedures to the apparatus.
12. Long-pass wavelength filters allow only light of wavelengths
greater than the indicated wavelength to pass through. Light of
Fluorescence-Based Equilibrium and Pre-Steady State Methods
283