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

Borries Demeler demeler at biochem.uthscsa.edu
Sun Sep 6 10:41:20 PDT 2009


Paul,

A couple of thoughts: You may have some aggregation in your equilibrium
experiment (perhaps because of the longer run time). Perhaps in the absorbance 
experiment you are clipping the higher OD's because they are out of 
the dynamic range of the optics, but in the interference experiment
the dynamic range is larger, and you are including the portion of the 
scan that correlates most with the aggregates. 

Another possibility is the presence of time invariant noise, but that 
should have been subtracted from the data by your baseline scan.

Finally, I wonder if the BeF3 refractive index is properly handled by
having a meniscus-matched BeF3 subtracted. I don't know the BeF3 density
off hand, but dependening on density it could for a gradient and 
significantly contribute to the refractive index signal. 

Others may have addtl. ideas...

-borries

> 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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