[RASMB] Sw Isothem Fitting in SEPHAT using Fluorescence Data

John J. Correia jcorreia at umc.edu
Thu May 3 13:27:09 PDT 2012


David

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.

From: David Chiovitti <david.chiovitti at gmail.com<mailto:david.chiovitti at gmail.com>>
Date: Thursday, May 3, 2012 3:17 PM
To: "rasmb at rasmb.bbri.org<mailto:rasmb at rasmb.bbri.org>" <rasmb at rasmb.bbri.org<mailto:rasmb at rasmb.bbri.org>>
Subject: [RASMB] Sw Isothem Fitting in SEPHAT using Fluorescence Data

Hi everyone,

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.

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.

Here is the isothem data:
0.82 0.1 3.6
2.74 0.1 3.48
7.4  0.1 3.50
22.7 0.1 4.0
66.6 0.1 4.94
200 0.1 4.90

The first value being the concentration of the protein, the second the concentration of the tRNA, and the third is the Sw value.

Other than SEDPHAT is there another program I could use to fit this Isotherm?

Thanks,
David Chiovitti

Samuel Lunefeld Research Institute
Mount Sinai Hospital Toronto


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