19. This fitting equation calculates the observed signal at a particular point in the titration from an observed experimental signal
for each protein species (free HEWL and bound HEWL) and
the mole fraction for that species. The observed experimental
signal for both species will be dependent on the total protein
concentration and the type and configuration of the plate
reader used. In the following figures, it was necessary to perform a simple normalization on fluorescence intensity data to
allow datasets from different instruments to be plotted
together. However, it is usually preferable to analyze directly
the non-normalized data in the original measurement units; for
example, this allows comparison of consistency of experimental
parameters between repeated runs on the same instrument
with the same settings.
20. All confidence intervals on K d presented in this chapter are
calculated by the method of error surface projection. For all
techniques except ITC, this was performed using Graphpad
Prism v7 (“asymmetric likelihood CI”) (https://www.gra
phpad.com/guides/prism/7/curve-fitting/index.htm?reg_
confidence_tab.htm). For ITC, this was performed in Sedphat.
21. The aim is to obtain a labeling stoichiometry of approximately
1 dye per HEWL molecule. To achieve this, a slight molar
excess (1.4-fold) of the NHS-activated dye over HEWL is
used in the labeling reaction. The pH and composition of the
labeling buffer were among those recommended by the dye
manufacturer. At pH 7.5, it is expected that both lysine sidechain amino groups and the N-terminus could be labeled,
raising the pH to around 8.5 would increase reactivity toward
the former, and lowering the pH to around 6.5 would selectively target the latter.
22. If it is much higher than 1:1, it is possible that the dialysis has
not proceeded to completion and there is considerable free dye
left in solution. This will significantly lower the signal-to-noise
in the final measurement.
23. This gives a final concentration of 150 nM HEWL-D488,
sufficient to give excellent signal-to-noise at moderate laser
intensities on the older instrument used for these measurements. Since the K d is in the low μM range, it is possible to
use a relatively high concentration or protein without limiting
the ability of the fit to discriminate K d . On newer instruments,
it should be possible to reduce this concentration significantly
to save material, if desired. For other interactions with K d in
the nM range, it is wise to use the lowest possible concentration of the constant species that affords acceptable signal-tonoise.
74
Xiaochun Li-Blatter et al.
for each protein species (free HEWL and bound HEWL) and
the mole fraction for that species. The observed experimental
signal for both species will be dependent on the total protein
concentration and the type and configuration of the plate
reader used. In the following figures, it was necessary to perform a simple normalization on fluorescence intensity data to
allow datasets from different instruments to be plotted
together. However, it is usually preferable to analyze directly
the non-normalized data in the original measurement units; for
example, this allows comparison of consistency of experimental
parameters between repeated runs on the same instrument
with the same settings.
20. All confidence intervals on K d presented in this chapter are
calculated by the method of error surface projection. For all
techniques except ITC, this was performed using Graphpad
Prism v7 (“asymmetric likelihood CI”) (https://www.gra
phpad.com/guides/prism/7/curve-fitting/index.htm?reg_
confidence_tab.htm). For ITC, this was performed in Sedphat.
21. The aim is to obtain a labeling stoichiometry of approximately
1 dye per HEWL molecule. To achieve this, a slight molar
excess (1.4-fold) of the NHS-activated dye over HEWL is
used in the labeling reaction. The pH and composition of the
labeling buffer were among those recommended by the dye
manufacturer. At pH 7.5, it is expected that both lysine sidechain amino groups and the N-terminus could be labeled,
raising the pH to around 8.5 would increase reactivity toward
the former, and lowering the pH to around 6.5 would selectively target the latter.
22. If it is much higher than 1:1, it is possible that the dialysis has
not proceeded to completion and there is considerable free dye
left in solution. This will significantly lower the signal-to-noise
in the final measurement.
23. This gives a final concentration of 150 nM HEWL-D488,
sufficient to give excellent signal-to-noise at moderate laser
intensities on the older instrument used for these measurements. Since the K d is in the low μM range, it is possible to
use a relatively high concentration or protein without limiting
the ability of the fit to discriminate K d . On newer instruments,
it should be possible to reduce this concentration significantly
to save material, if desired. For other interactions with K d in
the nM range, it is wise to use the lowest possible concentration of the constant species that affords acceptable signal-tonoise.
74
Xiaochun Li-Blatter et al.