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

Paul Leonard leonard at cabm.rutgers.edu
Sun Sep 6 09:52:53 PDT 2009


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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