Hello Everybody!
I am trying to understand how can I get the Kd from a curve generated from fluorescence assays. I made the experiments with proteins and two ligands, but I do not know how to analyse this result in a reliable way.
Hope you have something to help me =D
All the best,
Pamela
Plot the fluorescence intensity on the Y-axis and the concentration of the titrated (nonfluorescent) partner [L] on the X-axis. Use nonlinear regression to fit the data to the simple binding isotherm Y = C + Bmax[L]/(Kd + [L]), where Y is the fluorescence intensity, C is the fluorescence when the titrated ligand concentration [L] = 0, C + Bmax is the fluorescence when all the fluorescent partner is bound. If that gives a good fit, then stop. If not, then a more sophisticated approach may be needed. You may need to account for interference with the fluorescent measurement by the titrated partner, ligand depletion, nonspecific binding, and multiple binding sites with or without cooperative binding.
A good reference is the book Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis by Robert A. Copeland.
To corroborate Adam's response: From the plot of Fluorescence intensity against concentration of ligand, you can derive the slope of the graph as 1/Bmax; the y-intercept as Kd/Vm and x-intercept as -Kd.
Hanes-Woolf plot could be very useful for this also.
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