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<div>David</div>
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<div>You are entering total concentrations and signal average S values. You must write code that averages over the labeled species (its not Sw unless both are labeled with equal quantum yield) and then correct for free concentrations. Its a binomial solution
to the K expression: Y = Yo + deltaY*Fbound where Fbound is the binomial solution. Trivial actually. We do it in Scientist these days or in the old days in a Fitall software package.</div>
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<span style="font-weight:bold">From: </span>David Chiovitti <<a href="mailto:david.chiovitti@gmail.com">david.chiovitti@gmail.com</a>><br>
<span style="font-weight:bold">Date: </span>Thursday, May 3, 2012 3:17 PM<br>
<span style="font-weight:bold">To: </span>"<a href="mailto:rasmb@rasmb.bbri.org">rasmb@rasmb.bbri.org</a>" <<a href="mailto:rasmb@rasmb.bbri.org">rasmb@rasmb.bbri.org</a>><br>
<span style="font-weight:bold">Subject: </span>[RASMB] Sw Isothem Fitting in SEPHAT using Fluorescence Data<br>
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<div>Hi everyone,
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<div>I am studying a tRNA-protein binding interaction using the AVIV AU-FDS system. I am using a fixed amount of fluorescently labelled tRNA and titrating in the protein. From the c(s) plots I have determined that it is a slow exchange between the protein
and the tRNA. The tRNA give a peak at approximately 3.5 S. As I titrate in the protein the 3.5 S peak decreases and a new peak at 4.9 S appears. This is consistent with one tRNA being bound by one protein. For each titration I have used a weighted vbar
value based on the amount of protein and tRNA. I have been using the vbar value of 0.530 for the tRNA.</div>
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<div>For each of the titrations I have integrated the peaks to determine a weighted signal value and put them into a text file according to the formatting listed on the SEPHAT website. I open the file up with no issue in SEDPHAT and select the hetero-assocation
model. I enter the molecular weight and S values of each of the components and float the logKa value, which I set to 5. When I run and fit the data I get a curve that doesn't even come close to fitting the data points. I'm really at a loss to what I'm doing
wrong.</div>
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<div>Here is the isothem data:</div>
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<div>0.82 0.1 3.6</div>
<div>2.74 0.1 3.48</div>
<div>7.4 0.1 3.50</div>
<div>22.7 0.1 4.0</div>
<div>66.6 0.1 4.94</div>
<div>200 0.1 4.90</div>
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<div>The first value being the concentration of the protein, the second the concentration of the tRNA, and the third is the Sw value.</div>
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<div>Other than SEDPHAT is there another program I could use to fit this Isotherm?</div>
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<div>Thanks,</div>
<div>David Chiovitti</div>
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<div>Samuel Lunefeld Research Institute </div>
<div>Mount Sinai Hospital Toronto</div>
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