[RASMB] problem with fitting

[Walter Stafford]

Hi Marina,

      A couple of points:

First, gel filtration cannot give you a molar mass. It can give you  
only a Stokes radius.

Second SEDFIT does not have a monomer-hexamer model that can be used  
for direct fitting to a monomer-hexamer model.

If the material is pure and exhibits no concentration dependence you  
can use SEDFIT c(s) and C(M) to get the molecular weight.

However if it is reversibly associating/dissociating, you will need  
to use other software, like SEDANAL, in which you can specify a model  
of your choice.

Of course, you can use SEDANAL also to get the molar mass of non- 
interacting species as well without making any assumptions about  
frictional ratios.
	

Walter F. Stafford
Senior Scientist
Boston Biomedical Research Institute
64 Grove Street
Watertown, MA 02472
617-658-7808
stafford at bbri.org




On Mar 3, 2008, at 04:10, Fasolini, Marina [Nervianoms] wrote:

> Dear AUC users,
> I have a problem in the fitting of some data and I would like to  
> have your opinion.
>
> I want to control the sedimentation of a protein in order to define  
> the state of oligomerization. It is published as forming an   
> hexameric homo oligomer. From the crystal structure, it is a wheel  
> like structure of 160 angstrom  in diameter, with a central role of  
> 15 angstrom in diameter. The thickness is 20-40 angstrom. How can I  
> expect it to sediment? How can I improve my parameters or model in  
> order to improve the fitting?
>
> The protein is 50KDa  but in gel filtration it cames out as   
> hexamer (300 KDa). In my sedimentation experiment I see one peak  
> of  6.23S which corresponds to 140KDa. Fitting of the sedimentation  
> data was done with SedFit. Do you think I can say that it is a  
> trimer? I would expect it as an hexamer.
>
> I hope someone can help me.
>
> the conditions are :
> buffer 20mM Tris pH7, 150mM NaCl, 1mM DTT, 5%gly
> Vbar 0.7397
> Viscosity 1.02530
> Density 0.01183
>
> Thanks a lot
> Marina
>
>
>
> MARINA FASOLINI
> Structural Chemistry
> Nerviano Medical Sciences
> Viale Pasteur 10
> 20014 Nerviano - Milano
> marina.fasolini at nervianoms.com
> Tel. +390331581462
> Fax. +390331581360
>
> _______________________________________________
> RASMB mailing list
> RASMB at rasmb.bbri.org
> http://rasmb.bbri.org/mailman/listinfo/rasmb

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