OK to the sw picking mode then pick twice to define the svalues of the integration. In the sw isotherm constructor window, select save xp, save SEDPHAT, and fill the values of A and
B concentrations for each corrected c(s) distribution, the molar
extinction coefficient (xt component) of A and B, and values
for s A , s B , s AB , and log(K a ). Press Save, give a name to the file,
and save. In fact, 3 files with the same name but different
extensions will be created, the s w isotherm file (sw*.isotherm),
the SEDPHAT experiment file (sw*.xp), and the SEDPHAT
configuration file (sw*. sedphat).
4. Integrate all the superimposed corrected c(s) distributions to
generate EPT swfast isotherm file (isotherm of signal-weighted
s-value of the reaction boundary) (Fig. 4). In GUSSI, select
Integration/Make Isotherm/Hetero/EPT swfast, press OK to
the EPT swfast picking mode, then pick twice to define the svalues of the integration of the reaction boundary. In the EPT
swfast isotherm constructor window, follow the same procedure as for the sw isotherm constructor window to create
swfast* files.
5. Isotherm analysis in SEDPHAT (Fig. 4). Open SEDPHAT, in
Data/Load Experiment, open sw*.xp and swfast*.xp files,
enter the vbar value at 20
C, OK, then Data/Read Configuration from file, open sw*.sedphat. In the SEDPHAT A+B <->
AB model window that pops up, enter the molar masses for A
and B, and select the parameters s AB and log(K a ), OK. Choose
Run/Global Run, if the initial values in the model are not too
fare from the experimental data, Fit/Global Fit (see Note 9).
Then, in Global Parameters, select s A and s B , then Fit/Global
Fit. Evaluate the quality of the fit and the refined values for s A ,
s B , s AB , and logK a (see Note 10). Choose Statistics/Automatic
confidence interval, and search w projection method to calculate the confidence intervals of logK a . Press OK in the two
confidence search windows that pop up, then uncheck the
logK a parameter. Press CANCEL in the edit experimental
parameters window and OK in the constrained parameter window. In the confidence search window that pops up, enter the
P value of 0.95 and accept all the default parameters. At the end
of the calculations, a message box appears with the confidence
limits of logK a .
4 Notes
1. For example, the European Instruct-Eric (https://instructeric.eu/) and French FRISBI (http://frisbi.eu/) infrastructures provide open access to AUC instruments and expertise.
Heterogeneity and Affinity Interaction Analysis by Sedimentation Velocity
167
B concentrations for each corrected c(s) distribution, the molar
extinction coefficient (xt component) of A and B, and values
for s A , s B , s AB , and log(K a ). Press Save, give a name to the file,
and save. In fact, 3 files with the same name but different
extensions will be created, the s w isotherm file (sw*.isotherm),
the SEDPHAT experiment file (sw*.xp), and the SEDPHAT
configuration file (sw*. sedphat).
4. Integrate all the superimposed corrected c(s) distributions to
generate EPT swfast isotherm file (isotherm of signal-weighted
s-value of the reaction boundary) (Fig. 4). In GUSSI, select
Integration/Make Isotherm/Hetero/EPT swfast, press OK to
the EPT swfast picking mode, then pick twice to define the svalues of the integration of the reaction boundary. In the EPT
swfast isotherm constructor window, follow the same procedure as for the sw isotherm constructor window to create
swfast* files.
5. Isotherm analysis in SEDPHAT (Fig. 4). Open SEDPHAT, in
Data/Load Experiment, open sw*.xp and swfast*.xp files,
enter the vbar value at 20
C, OK, then Data/Read Configuration from file, open sw*.sedphat. In the SEDPHAT A+B <->
AB model window that pops up, enter the molar masses for A
and B, and select the parameters s AB and log(K a ), OK. Choose
Run/Global Run, if the initial values in the model are not too
fare from the experimental data, Fit/Global Fit (see Note 9).
Then, in Global Parameters, select s A and s B , then Fit/Global
Fit. Evaluate the quality of the fit and the refined values for s A ,
s B , s AB , and logK a (see Note 10). Choose Statistics/Automatic
confidence interval, and search w projection method to calculate the confidence intervals of logK a . Press OK in the two
confidence search windows that pop up, then uncheck the
logK a parameter. Press CANCEL in the edit experimental
parameters window and OK in the constrained parameter window. In the confidence search window that pops up, enter the
P value of 0.95 and accept all the default parameters. At the end
of the calculations, a message box appears with the confidence
limits of logK a .
4 Notes
1. For example, the European Instruct-Eric (https://instructeric.eu/) and French FRISBI (http://frisbi.eu/) infrastructures provide open access to AUC instruments and expertise.
Heterogeneity and Affinity Interaction Analysis by Sedimentation Velocity
167