Hi, I have done fluorescence experiment for seeing interaction of purified protein with the oligo. Now I need to calculate stoichiometry and Kd. I have made all the graphs. Also, how can binding sites be calculated by fitting values in to the formula for fluorescence of protein-DNA binding data?
How you analyze the data depends on the details of the experiment. Was the experiment done under tight binding conditions, i.e. the fluorescent reporter concentration was close to or above the Kd? If so, you should use the quadratic formula for the binding equilibrium, rather than the Langmuir equation. Also, in the tight binding situation, you should be able to determine the binding stoichiometry directly by seeing at what concentration of the binding partner the binding-induced fluorescence change is complete, extrapolating the linear part of the titration curve.
A potential problem with the tight binding experiment may be that the there isn't enough curvature in the titration plot to calculate the Kd accurately. Reducing the reporter concentration may solve the problem, but you can also do an experiment, if feasible, in which the fluorescent reporter concentration is far below (no more than 5%) the Kd, and calculate the Kd using the Langmuir equation. However, in this experiment you may not be able to measure the stoichiometry. If there is cooperativity of binding, you will have to use a more complex equation, such as the Hill equation. If there are multiple non-cooperative binding sites with different affinities, you will have to use a multiple Langmuir-type equation.
Thank you so much Adam B Shapiro for your response. Its really of great help.
What kind of experiment did you perform? What are the units of the abscissa and ordinate in the graphs you've plotted?
Hi Priya,
There is a very useful software package called DynaFit. It can be downloaded from www.biokin.com. It is free but you should request an academic license, Dr Kuzmic sends it to you within a few days.
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