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Hi all-<br>
For equilibrium work, the upper limit is set by the optical focus and
the concentration (refractive index) gradients. The absorbance system <u>cannot</u>
handle nearly as steep a concentration gradient as the interference
optics can for the reasons pointed out by Arthur. So you should use
interference optics. <br>
If you use shorter pathlength cells, you must refocus the optics to
avoid the problems Arthur pointed out. The hassle of refocusing
prevents most people from attempting it.It can be done, though.<br>
The bigger problem is not optics, focus or wavelength, but proper data
interpretation. Nonideality is not dependent on the optical system, but
is a property of the chemical system. Current data analysis programs
use a single nonideality coefficient to describe the interactions that
lead to nonideal solution behavior. At the concentrations where you
wish to work, it is not likely that any of the programs will fit the
data properly. You could use scaled particle theory to fit a single
parameter (the effective radius) to a power series. It is not clear how
to interpret the coefficients of the power series.<br>
Alternatively, you could fit for the average apparent molecular weight
as a function of cell loading concentration and determine the activity
coefficient at several protein loading concentrations. Activity
coefficients greater than one indicate repulsive non-ideality. Activity
coefficients less than one indicate associative nonidealitiy. You get
one determination per sample (unless you use a program like Biospin or
MStar to determine the point-average apparent molecular weights). <br>
Determining the activity coefficient is very, very quick (especially
the data analysis) and thermodynamically rigorous. The rub is in the
interpretation of the data. Associative behavior (e.g. under NMR
conditions) may or may not be because of protein-protein interaction
(e.g. dimer formation). Reduced activity coefficients may result from
close-range favorable solute-solute weak-forces (e.g. dipole-dipole
interactions, solute-solute solvation) or may result from
less-favorable solute-solvent (or solvent-solvent) interactions at high
concentrations. <br>
The problem is not due to limitations of AUC, but to the model
("cartoon") biochemists and molecular biologists use when thinking
about high concentration solutions. A different method will provide the
same sort of interpretative ambiguity. It may be worthwhile to
interpret high-concentration data using a Van der Waals model that
contains a single attractive and single repulsive coefficient.<br>
Best wishes,<br>
Tom<br>
<br>
<br>
<br>
<br>
Beld, Joris wrote:
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<p class="MsoNormal">Dear all,<o:p></o:p></p>
<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal">A colleague asked me whether analytical
ultracentrifugation
has an upper limit with regard to the concentration of the protein.
They want
to measure the protein at the same concentration as the NMR experiments
(> 1
mM). I am not entirely sure but I thought this should be no problem.
One could
easily measure off-peak at another wavelength than 230nm, e.g. 235nm or
280nm,
right?! Or does one run into non-ideality phenomena when doing
sedimentation equilibrium
at these high protein concentrations?!<o:p></o:p></p>
<p class="MsoNormal">Thanks a lot in advance for any feedback.<o:p></o:p></p>
<p class="MsoNormal">Best wishes,<o:p></o:p></p>
<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal">Joris Beld<o:p></o:p></p>
<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal">Hilvert Group<o:p></o:p></p>
<p class="MsoNormal">ETH Zürich<o:p></o:p></p>
<p class="MsoNormal">Switzerland<o:p></o:p></p>
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