[RASMB] Sedfit peak integration

[John Correia]

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
2500 North State Street
Jackson, MS  39216
(601) 984-1522                                 
fax (601) 984-1501                             
email address: jcorreia at biochem.umsmed.edu     
homepage location: http://biochemistry.umc.edu/correia.html 
dept homepage location:    http://biochemistry.umc.edu/ 
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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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