I'm performing Sypro Orange fluorescence-based thermal shift assays of my mutant reductase protein (HsPYCR2_R251C) in presence and absence of its substrate and product ligands, using a MyiQ Single-Color Real-Time PCR Detection System with optics module instrument & iQ5 Optical System software (Bio-Rad, version 2.1). The Melt Curve data output yields both (RFU vs. T) data and (-d(RFU)/dT vs. T) data. After using MS Excel 2013 to manually subtract out blanks' fluorescence values from my sample values from my 4 technical replicates of each protein condition, I input that data into SigmaPlot 12.0. I've already been able to fit the (RFU vs. T) data with a user-defined Boltzmann equation that I customized as f=y0+((a-y0)/(1+exp(-(x-x0)/b))), where a=maximum y-value, y0=minimum y-value, x0= Tm value of interest, & b=slope factor. Now, my goal is to have the REPORT file of the graphical data fit tell me the parameter estimate for x0 = Tm value of interest as (value +/- std. error) for my publication of these results. However, in some cases when I cannot visually fit the Boltzmann equation to the attached (RFU vs. T) data plot, BUT it is clearly visible that the (-d(RFU)/dT vs. T) data shows a conspicuous local minimum "peak" y-value that is the Tm value of interest, however I am having trouble with the syntax for a user-defined graphical equation that'll fit this data and give a REPORT file of the parameter estimate for x0 = Tm value of interest as (value +/- std. error). I've tried using an equation as the negative first derivative with respect to T, of my user-defined Boltzmann equation, as f = (y0-a)/((1/exp((x0-x/b))) + 2 + (exp((x0-x)/b))) but I'm encounter parameter estimates for x0 that are inaccurate and not matching what the attached (-d(RFU)/dT vs. T) data plot visually shows.
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