[RASMB] problem with fitting

[Tom Laue]

Hi-
A small point- gel filtration does not provide a Stokes radius. 
Experiments suggest that gel filtration provides the "projected radius" 
corresponding to the radius of the smallest shadow a structure can cast. 
This projected radius is obtained, however, in the limit of zero flow 
(e.g. using the equilibrium methods developed by Gary Ackers' group). 
For a finite flow rate, the apparent radius will be tend to move towards 
the projected radius corresponding to the the radius of the largest 
shadow a structure can cast. Hence, elongated molecules (at finite flow 
rates) will come out at an apparent radius that is too large. Neither 
projected radius is related to the Stokes radius.
Best wishes,
Tom

Walter Stafford wrote:
> 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 <mailto: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 <http://www.nervianoms.com/>
>> Viale Pasteur 10
>> 20014 Nerviano - Milano
>> marina.fasolini at nervianoms.com <mailto:marina.fasolini at nervianoms.com>
>> Tel. +390331581462
>> Fax. +390331581360
>>  
>> _______________________________________________
>> RASMB mailing list
>> RASMB at rasmb.bbri.org <mailto:RASMB at rasmb.bbri.org>
>> http://rasmb.bbri.org/mailman/listinfo/rasmb
>
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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
www.bitc.unh.edu
www.camis.unh.edu