[RASMB] FDS data fitting in Sedfit

[Tom Laue]

Hi Steve-

So long as the quantum yield is constant, you may use the integral to 
determine the amount of signal (material) contained in a range of s 
values (e.g.over a peak). If you want the fractional amount, you have 
the concern of separating the GFP signal from the autofluorescent 
signal. To do this, you would have to integrate those s-ranges that 
contain only the GFP signal (and have some way of knowing what those 
ranges are). Presumably, your non-GFP controls should provide an idea of 
those s ranges to not include in your analysis. Then divide the area for 
any given range by the sum of the signals from all of the ranges 
included in your analysis to get the fractional amount.

Autofluorescence is a particular problem in yeast, though there also may 
be oxidation products in serum (thought to be principally bilirubin) 
which sediment with albumin. We have not encountered significant 
autofluorescence in E. coli extracts up to this time.

Yeast are of particular interest for FDS since every gene in S. cervisae 
has had GFP added to it. We are doing a fair number of experiments 
involving yeast GFP constructs and their complexes in cytosol and 
obtaining very interesting insights into the regulation of genes and the 
variation of complex size with culture conditions, cell cycle, etc. With 
respect to yeast autofluorescence, in addition to the 4 s boundary 
(believed to be the riboflavin protein, which is abundant in yeast), 
there also can be a 40 s boundary which is thought to be the consequence 
of a heme or flavin associated with the small ribosomal subunit. I have 
a summer student working on understanding and controlling (hopefully) 
the autofluorescence in yeast.

Best wishes,
Tom

smcbryan wrote:
> I have recently collected SV data from clarified, whole-cell yeast extracts
> which express GFP-tagged proteins of interest. Along with two of these
> samples, I subjected the buffer and a non-GFP-expressing extract to SV. As
> documented, there are 'autofluorescent' species in yeast extracts, in
> particular a 4S (60 kDa) riboflavin binding protein which is complicating
> my
> analysis of the results. This species should show up in all three extract
> samples, which is does not.
>  I have the following question: does amplitude of the value on the Y-axis
> from a c(s) fit
> represent the fraction of each component (as seen on the X-axis), based
> upon
> its' contribution to the total signal from the sample, or does the peak
> need to be integrated. 
> thus, can I divide
> each components c(s) into the total sedimenting fluorescent signal from the
> sample to determine its' numerical fraction of the sample (ignoring
> possible
> quantum yield and quenching effects)?
> thanks for any input. 
> steve
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>   

-- 
Department of Biochemistry and Molecular Biology
University of New Hampshire
Durham, NH 03824-3544
Phone: 603-862-2459
FAX:   603-862-0031
E-mail: Tom.Laue at unh.edu
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