[RASMB] Sedfit peak integration
Walter Stafford
stafford at bbri.org
Fri May 23 11:14:58 PDT 2008
Hi Dean,
By slow I mean an off rate slower than 1e-5 sec^-1 or slower. An
off rate faster than 1e-4 sec^-1 will distort the boundary enough to be
noticeable. And any thing faster than about 0.01 will behave essentially
as instantaneous at the usual speeds in the centrifuge.
Hope that helps,
Walter
Dean Malencik wrote:
> Walter,
>
> You stated
> "Just remember that for an interacting system - unless it's
>
>> extremely slow - the areas under the peaks are meaningless..
>>
>
> When you say slow what kind of times are we talking about?
>
> Dean Malencik
> malencid at ucs.orst.edu
>
> *********** REPLY SEPARATOR ***********
>
> On 5/20/2008 at 1:09 PM Walter Stafford wrote:
>
>
>> Hi John,
>>
>> To add the what John said: SEDANAL can also treat kinetically controlled
>> systems as well with forward and reverse rate constants as fitting
>> parameters. Just remember that for an interacting system - unless it's
>> extremely slow - the areas under the peaks are meaningless; in fact, the
>> whole concept of peaks that correspond to species is meaningless. Those
>> data must be fit to an interacting system model that can correctly
>> represent the reaction boundary shape and evolution with time. Only a
>> single weight average sedimentation coefficient value can be obtained
>>
> >from each loading concentration (i.e. each cell loading). Whole boundary
>
>> global fitting to multiple data sets gives you much more data (i.e.
>> better signal-to-noise) to work with and much better discrimination
>> between potential association schemes.
>>
>> Walter
>>
>> John Philo wrote:
>>
>>> 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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>> --
>> Walter F. Stafford, Ph.D.
>> Senior Scientist
>> Analytical Ultracentrifugation Research Laboratory
>> Boston Biomedical Research Institute
>> 64 Grove Street
>> Watertown, MA 02472-2829
>> -------------------------
>> mailto:stafford at bbri.org
>> direct dial: 617-658-7808
>> receptionist: 617-658-7700
>> -----
>> Boston Biomedical Research Institute...
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>
>
>
>
>
--
Walter F. Stafford, Ph.D.
Senior Scientist
Analytical Ultracentrifugation Research Laboratory
Boston Biomedical Research Institute
64 Grove Street
Watertown, MA 02472-2829
-------------------------
mailto:stafford at bbri.org
direct dial: 617-658-7808
receptionist: 617-658-7700
-----
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Please visit us at http://www.bbri.org/
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