[RASMB] Sedfit peak integration

John Philo jphilo at mailway.com
Tue May 20 09:22:58 PDT 2008


Tom,
 
I agree with Jack Correia's points but I am not sure he stated forcefully
enough that the heights of the peaks are irrelevant---it is only the areas
that are potentially meaningful. The peak widths from the c(s) method depend
on signal/noise ratio, maximum entropy settings, etc. and in general have no
direct physical meaning, and therefore the same is true for the peak
heights. 
 
I'm also not sure you and Jack are using the term "normalization" in the
same sense. The normalization Jack is referring to sets the total area under
the curve of a g(s*) or c(s) distribution to 1 (100%) so the area for each
peak gives the fraction of that species. You can manually normalize a c(s)
distribution from SEDFIT by exporting it and dividing the Y values by the
total area reported by SEDFIT's integration function; in DCDT+ normalization
is a built-in option.
 
With regard to fitting SV data to an explicit competition model, since
SEDANAL allows developing and fitting your own assembly models I am sure it
could do this.
 
John

  _____  

From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On
Behalf Of Thomas Jowitt
Sent: Tuesday, May 20, 2008 8:55 AM
To: John Correia; rasmb at rasmb.bbri.org
Subject: RE: [RASMB] Sedfit peak integration



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.

 

 

 

-------------------------------------------------------------------
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/
-------------------------------------------------------------------




>>> 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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