[RASMB] Re: RASMB digest, Vol 1 #319 - 5 msgs

[medakachou]

> Date: Tue, 13 Jul 2004 10:59:00 -0500
> From: "John Correia" <jcorreia at biochem.umsmed.edu>
> To: <jphilo at mailway.com>, <r6243023 at ms48.hinet.net>,
>    <arthur.rowe at nottingham.ac.uk>, <rasmb at server1.bbri.org>
> Subject: Re: [RASMB] difference of p = 0.95, 0.68 and 0.55, the
> confidencelevel in the sedfit c(s) distributi
>
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> One problem I have with these discussions is they are method of analysis
> focused and until Arthur's illustion to "know your system" not focused
> on the molecules, the mechanism, the interactions.  Were we just talking
> about one run and one concentration or a series of conectrations?  Why
> do you want to integrate?  Are the "peak" positions constant or do they
> change with concentration?  Is this in fact an impure system of
> nointeracting species or aggregates, or are these aggregates of a single
> component?  Is there any reversible interaction going on?
>
> The goal is to describe your system in molecular and mechanistic terms
> and then fit the data individually & globally to that model to prove the
> hypothesized mechanism.  Statistics, assumptions, simulations are all
> important.  Now what is going on in your system?

Dear John,

    Yes, I should give more information about my case. I expressed and
purified a 34 kDa protein and it's N-terminal or C-terminal truncated
fragments by E. coli expression system. The protein purity is > 99% by
SDS-PAGE.  This protein has two isoforms. I study them in three different
concentration: 0.15, 0.50 and 1.00 mg/ml in PBS (pH7.3). By using
sedimentation velocity and c(s) distribution analysis, I found one isoform's
N-terminal truncated fragments showed a 10 "peaks" pattern whose s is from 3
to 23 at 1.00 mg/ml. While at 0.15 mg/ml, only 5 peaks were found at s = 3
to 12. It means it should be a single component aggregation and is
concentration-dependent. The other isoform didnot show this characteristics.
I want to give my paper some quantitative data about this difference, so I
chose "Origin peak fitting module" and analyzed the pattern of gaussian
peaks. The reviewer thought it is overinterpretation (about the
fused-"peaks") and suggested me lowering the cinfidence level to p = 0.7.
I've tried and found the resolution is better (every "peaks" is still
existed). These two days I've tried Jack Lebowitz's comment and gained some
quantitative data. I'm going to compare them and hope it can make my paper
more quantitative sound.
    By the way, while I isolated the major "peak" species by using
gel-filtration chromatography (S-300 column) and concentrate them (I need
higher concentration), It just change back to the same "multi-peaks"
situation (by sedimentation velocity). I think it's not a non-interacting
but a associating system, right? Thanks your help.

Chi-Yuan Chou
PhD student, the Institutes of Life sciences, National Defense Medical
Center, Taipei, Taiwan
e-mail: r6243023 at yahoo.com.tw