[RASMB] Frict ratios and sedfit

[Jack Kornblatt]

Samantha Jones (PRION) wrote:

> Hello
>
> I recently analyzed some data using c(S) distribution model on Sedfit. 
> The distribution showed a few peaks, with the main peak being at 4S. I 
> floated the frictional ratio and came back with approx 1.5. I then 
> converted this to a c(M) distribution with the main peak now showing 
> approx 65,000 Daltons. Knowing our monomer weight as 16,400 Daltons I 
> concluded this was a tetramer.
>
> At the same time the same protein sample was analyzed using 
> synchrotron X ray scattering. Their results concluded an octomer, a 
> two ring structure.
>
> With this knowledge I went back to the data and reanalyzed using 
> Sedfit, c(s) and c(M) again. Using the calculator function on Sedfit I 
> calculated that for 4S with a frictional ratio 1.5 this would be 
> roughly the tetramer weight, however, a frictional ratio of 2.4 for 4s 
> gives a molecular weight equal to the octomer. When I entered this 
> into the parameters of the c(M) model the rsmd values show the f/f0 of 
> 2.4 gives a better fit, 0.07 to 0.119 for f/f0 of 1.5.
>
> My question is how I could have got this answer without the knowledge 
> of the scattering data? Is the use of the calculator function to fit 
> data recommended? Especially in a sample with three peaks?
>
>  
>
> Many thanks
>
>  
>
> Samantha
>
>  
>
> Samantha Jones
>
> MRC Prion Unit
>
> Institute of Neurology
>
> Queen Square
>
> London
>
> WC1N 3BG
>
>  
>
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Hello Samantha.
My understanding of sedfit is that when you have a mixture of  molecular 
species in your AUC sample, the frictional ratio becomes very hard to 
estimate for any one of the particular species.
There are other techniques that might be better suited to evaluate the 
molecular size of the individual species. You might consider two 
dimensional gel electrophoresis using a gradient native gel in the first 
dimension and an SDS gel in the second. The peak intensities and the 
"molecular sizes" can be estimated to +/- 10% if you run standards at 
the same time.
all the best
jack kornblatt
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