[RASMB] Why is my interference data different from teh absorbance data

[Arthur Rowe]

Hi Paul

What software are you using, I wonder? And how are you doing the  
experiments? It is a curious fact that getting molecular weight values  
by SE using interference optics is not actually the easiest of things  
to do. You have to float the baseline, which is a parameter rather much  
correlated with the sigma parameter!

On the basis of the old curve-fitter's saying - "fix what you know,  
float what you don't" - I would normally myself treat sigma as  
something given to me by SEDNTERP and therefore fixed, leaving the  
interaction parameter to be floated. A single SE run will not  
distinguish between dimer being present in equilibrium and dimer being  
present as a mixture, for that you need several runs with c varied.  
Analysed globally or not, this will sort it out.

Regards to you and all

Arthur


On Sep 6, 2009, at 17:52, Paul Leonard wrote:

> Dear all,
>
> I have been running both sedimentation velocity and sedimentation
> equilibrium AUC experiments on a relatively simple protein system.
>
> The sedimentation velocity experiments at multiple protein  
> concentrations
> show that the protein is just a monomer unless BeF3- is present (to  
> mimic
> phosphorylation) and then all of the protein becomes dimeric.
>
> However if I run sedimentation equilibrium experiments at multiple  
> speeds
> I find that if I use the absorbance optics it is in agreement with the
> sedimentation velocity result - protein is just a monomer in the  
> absence
> of BeF3- but fits as a dimer if BeF3- is present.
>
> However if I look at the interference scans for the same cells (for
> sedimentation equilibrium AUC) the protein looks considerably bigger -
> i.e. it would appear that it is in equilibrium between monomer and  
> dimer
> in the absence of BeF3-
>
> I have also record analytical size exclusion chromatography profiles  
> for
> concentrations of the protein all the way from 10uM upto 500 uM and if
> BeF3- is absent I cannot see any evidence of dimer formation.  If I add
> BeF3- the protein is all dimer.
>
> Therefore I am pretty certain that I should only be seeing monomer in  
> the
> absence of BeF3- and dimer if BeF3- is present so I'd like to know why  
> my
> interference data makes the protein look larger than it should.  Is  
> there
> some correction that I need to apply.  I have recorded water blanks at
> each speed and it makes virtually no difference if I subtract these  
> scans
> (Not enough of a correction to account for the additional curvature in  
> the
> scans).
>
> Could it be that the radial calibration is wrong?  Is something wrong  
> with
> the interference optics?  Any explanation would be greatly appreciated.
>
> I fear that if we cannot get reliable results for this simple case  
> using
> this equipment for more complex systems will become impossible (at  
> least
> for sedimentation equilibrium experiments).
>
> Thank-you for any help you can provide.
>
> Paul
>
>
>
>
>
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>
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Arthur J Rowe
Professor of Biomolecular Technology / Director NCMH Business Centre
School of Biosciences
University of Nottingham
Sutton Bonington
Leics LE12 5RD

TEL:  0115 9516156
FAX:  0115 0516157
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