[dtop]

So I'm trying to find the association constant for ethidium bromide bound to DNA using fluorescence techniques.  I've acquired all my data and it qualitatively matches well with the literature values.  The problem I'm having is doing the math to extrapolate various DNA binding / Fluorescence parameters.

If anyone has any experience with this to give some aid:
So what I've done is collect data, where I keep constant the amount of ethidium bromide and I vary the amount of DNA in various solutions.
So I know two things:

F = F⁰ (C_t - C_b) + F^bC_b

Where F = observed fluorescence at a given DNA concentration
F⁰ = Fluorescence of free ligand [ethidium bromide]
F^b = Fluorescence if the bound ligand to DNA

Also, I am given that:

KC_b² - C_b (KD_t + KC_t +1) +KD_tC_t = 0

Where
C_t = total ethidium bromide concentration
C_b = concentration of ethidium bromide that is bound to DNA
D_t = total DNA concentration
K = DNA Association constant

Now the literature I am following goes on to say the following:   "A plot of F versus D_t at fixed C_t allows the determination of values for K, F₀, and F_b."  The paper it references says "Data in the form of fluorescence response F as a function of total DNA site concentration at fixed concentration of ligand can then be fit by nonlinear least squares methods to get K, Fb, and F0."  Ive read about 5 papers, and every one of them just points to some specific software package that performs the fit but does not go into detail of how the fit is performed.

I guess my question is this:  How do I use a nonlinear least squares method to solve for those parameters?  I've tried using MATLab and OriginPro to do this, but I am at a loss as I dont quite know how to incorporate the above equations into the fitting algorithms of the software.  I'd prefer to use MATLab as I only have a trial of OriginPro

Additionall, can anyone point to any specific reference papers that will clarify this methodology?

Reference paper:  http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1304907/

[tanti]

I have similar problem. Did you find any solution?