[RASMB] SV on membrane proteins

[Mark Agacan]

Hello,
 
I'm also running SV on a membrane protein in 150 mM NaCl, 50 mM NaCl,
and 0.1 % Foscholine-12.
 
I have no data for the detergent density or viscosity and so I tried to
omit it completely from the input parameters and see what sedfit c(s)
would output.  
 
The mass values were way off from what was expected... as expected.
 
Can anyone suggest a way I can deal with density, viscosity, vbar
(without actually measuring the density) for this buffer / detergent
combination?
 
Cheers
 
Mark
 
 
 
 
 
 
 
 
 
 
 
 
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Dr Mark Agacan, Scientific Officer for the Division of Biological
Chemistry and Drug Discovery,
Wellcome Trust Biocentre, College of Life Sciences, University of
Dundee, Dundee, DD1 5EH 
Tel: +44 1382 386095    Fax: +44 1382 345764    Mobile: 07525 451 117
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>>> Chad Brautigam <chad.brautigam at utsouthwestern.edu> 5/13/2010 15:41
>>>
Greetings, All, from the newbiest of membrane-protein newbies,

First, let me thank all of you experts who answered (both on- and off-

board) my last question on the topic of membrane proteins.  'Preciate 

ya, as we say here in Texas.

I recently did some SV experiments on a membrane protein solubilized  
in the accursed floating detergent LDAO (DDAO to some of you).  I was 

able to fit the data nicely in SEDFIT using a discrete species model.  

By using the trick of setting the vbar to 0, I fitted for s and for  
the buoyant molar mass (Mb).  Hooray.

In a separate experiment, I used a combination of interference optics 

and absorbance optics to measure the amount of detergent bound to the 

protein.  This was very consistent from sample to sample (I tried 2  
different protein concentrations).  This method is à la le Maire et al 

(2008) Nat. Protocols, vol. 3, 1782.

OK, so I have Mb and I know the amount of detergent bound to the  
protein on a g/g basis (this quantity is deltasubD).

We have the justly famous formula:
Mp = Mb (1-phiprime*rho),
where (neglecting bound lipid)
(1 - phiprime*rho) = (1 - vbarp*rho) + deltasubD(1-vbarD*rho)

So, since I have Mb and deltasubD, and I know rho and the vbars, can I 

just plug the Mb that I get from SV into these equations and get a  
good estimate for Mp, or is there some SV subtlety here that my newbie 

brain hasn't apprehended?  I know this can be done for SE, but I've  
never seen anyone attempt it for SV.

Any and all responses will be 'preciated.

Chad

======================================
Chad A. Brautigam, Ph.D.
Assistant Professor
Department of Biochemistry
The University of Texas
Southwestern Medical Center at Dallas
Department of Biochemistry
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
Office:  214-645-6384
Fax:  214-645-6353
Email:  chad.brautigam at utsouthwestern.edu





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