[RASMB] Sedfit peak integration

[John Correia]

If you could use different wavelengths during the same run to quantify different components you would need to collect data at peaks or minima to avoid bouncing in the abs values between scans.
 
Sedanal allows you to build any model you can conceive of for fitting velocity or equil data.  So a 2A <-> A2 plus an A + I <-> AI model with or without slow kinetics is easily constructed for velocity data.  Independent knowledge of K's would help for uncolored components, but in principle you can analyze it this way.  Best to contact Walter Stafford for getting it up and running in your facility.  Good luck.
 
& I just saw John Philo's comments & agree its critical to use areas under the curves.  Sedanal also does g(s) and normalization but using an integrate function in Sedfit and normalizing later should also work.  Comparing zones in this way, especially from different wavelength data, can reveal interesting heterogeneity in complex systems as well.
 


>>> On 5/20/2008 at 10:54 AM, in message <20080520165443078.00000001524 at ThomasJowitt>, "Thomas Jowitt" <thomas.a.jowitt at manchester.ac.uk> wrote:

Thanks John
This is a slowly interacting system that we have characterized using the kds from sed eq. however it is complicated by the fact that it is partially calcium dependant, although they do interact slowly when no calcium is present (at least non-added). I will have a look at the DCDT plots and try to normalize them there. We are attempting to compete with fragments of the same molecule to establish which domains are responsible for dimerisation, therefore no color unfortunately. You are right in that the concentration of competition fragments is critical and we can see competition with some of the fragments. Only quantifying between different runs at different wavelengths is tricky. 
I am not familiar with competitor models for direct boundary fitting.
Tom
 


From:John Correia [mailto:jcorreia at biochem.umsmed.edu] 
Sent: 20 May 2008 15:34
To: thomas.a.jowitt at manchester.ac.uk; rasmb at rasmb.bbri.org 
Subject: Re: [RASMB] Sedfit peak integration

 

Tom

 

You described a noninteracting or slowly interacting system.  A reversible monomer dimer system is one peak, skewed near the mid point of the transition.  For a reversible system plot Sw vs total concentrations to observe the extent of dimerization.  This requires a Kd in the accessible concentration regime.   If you see two peaks its nonreversible or slow kinetics, possibly disulfide crosslinking.  Tight competitive binding with an inhibitor might produce a free monomer zone but the expected shape or resolution into two zones will depend upon the relative affinity of competitor with Kd for dimerization.  A simple assumption in your analysis may be too simple.  Direct boundary fitting to a competitive model is also possible.  Does the inhibitor have color?  Knowing inhibitor concentration is a critical feature for this analysis.  Normalization of the peaks often makes it easier to see the transition or changes in boundary shape but this is a qualitative analysis.  Use the total area under the curves to normalize.  Some programs like DCDT+ have a normalization function built in.

 

 

 

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Dr. John J. "Jack" Correia
Department of Biochemistry
University of Mississippi Medical Center
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>>> On 5/20/2008 at 9:05 AM, in message <20080520150555687.00000003664 at ThomasJowitt>, "Thomas Jowitt" <thomas.a.jowitt at manchester.ac.uk> wrote:

Hello
 
I have a question regarding peak integration between different sedfit analyses. In an effort to gauge competition in a self-associating system, velocity was used to assess the level of dimerisation and subsequent competition using different concentrations of dimer and the competing element. The question is one of assessing the relative degree of competition, therefore the integration or relative size of the peaks. Is it right to normalize the different runs to the height of the dimer (predominant species), and therefore compare the relative size of the monomer peaks for competition, and if so can the integrated value of the normalized peaks be used also, or is that more subjective?
 
Thanks for any thoughts
 
Tom Jowitt


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