[RASMB] Sedfit peak integration

[Thomas Jowitt]

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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 try integrating the area under DCDT curves

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 maybe too simple. Direct boundaryfitting 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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