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Hi Fumio-<br>
John is right about the Weiner skewing and I agree that using thinner
cells is a good idea. <br>
I believe the jogs in your data may have a slightly different origin.
At each radial position the software can only determine the fractional
fringe shift (relative to the first radial position, defined as 0
fringe shift). In order to keep track of the total fringe shift, the
software accumulates the total fringe shift, by adding or subtracting
the increment between the current calculation and the preceding
fractional fringe determination. There is a limit to the maximum fringe
shift between adjacent points that can be accounted for. If the data
are steeper than that, i.e. the fringes shift by 0.9 fringe between two
radial positions (or in cases where the fringes are of poor quality)
the software gets confused and you get the sorts of jumps seen in your
data. In general, the software-caused jumps occur at concentration
gradients that are below the Weiner skewing limit.<br>
<br>
Beckman sells 3 mm centerpieces and Spin Analytical sells 3 mm and 1.2
mm centerpieces. The spacers for these centerpieces place the solution
approximately at the midpoint of the cell. In principle, the
interference optics should be focused at the 2/3 plane of the solution
to minimize the effects of Weiner skewing. Fortunately, the shorter
optical path reduces the effects if Weiner skewing (see Yphantis, DA
[1964] Biochemistry 3:297-317, for a full discussion). There is not a
way to focus the "stock" XLA interference optics with sufficient
accuracy. Jeff Lary has modified the XLA lens mounts with micrometer
screws, which do have sufficient accuracy (though using Gropper's
spurs, the preferred method of determining the proper focus, is not
available on the XLA). <br>
<br>
John's second point also is true. Most current software have limited
capabilities to handle nonideality in sedimentation velocity data. This
means that Weiner skewing has a relatively small effect on boundary
shape compared to nonideality. SedFit/SedPhat uses two proportionality
constants to account for the effects of thermodynamic and hydrodynamic
nonideality. Note that these two terms have opposite effects on
boundary shape- hydrodynamic nonideality sharpens boundaries, repulsive
thermodynamic nonideality broadens them. For symmetrical, globular
proteins with low to moderate charge and in solvents having an ionic
strength ~0.1 M, the model seems to work adequately for a single
component up to ~15 - 20 mg/ml (hydrodynamic nonideality dominates).
The models cannot handle systems that have significant concentrations
of multiple components (separate cross terms are needed that reference
the individual concentrations, e.g. the Johnston-Ogston effect) or
systems where both attractive and repulsive thermodynamic terms are
significant. Walter Stafford is working adding the ability to handle
these cross terms to Sedanal.<br>
<br>
With the Aviv fluorescence detection system, it is possible to acquire
data in very complex, very concentrated solutions (e.g. serum, sputum,
urine, cell lysates) that exhibit all sorts of nonideality. For systems
like serum, etc. while the nonideality is complicated, it also is
relatively constant from sample to sample, and you often can answer
some very interesting qualitative questions deductively. <br>
<br>
Best wishes,<br>
Tom<br>
<br>
<br>
John Philo wrote:
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<p><font size="2">Fumio,<br>
<br>
There is a limit on the maximum gradient that can be measured
(fringes/mm) due to Weiner (? spelling) skewing, and you are exceeding
that limit. As Ariel said you should switch to 3 mm centerpieces, and
then at some point to get to higher concentrations you would have to
drop the rotor speed too to broaden the boundaries. If you look through
the old RASMB postings you will find a number of excellent posts from
others about Weiner skewing and proper optical alignment to minimize
distortion of strong gradients.<br>
<br>
But probably the more important question is what are you going to be
able to do with such data even if you collect it? Remember, in velocity
there is hydrodynamic non-ideality as well as thermodynamic
non-ideality, so the non-ideality effects will kill you at much lower
concentrations than in equilibrium. To my knowledge the only model
available to analyze a velocity experiment at 10 mg/mL is a single
non-ideal species. At high concentrations you cannot even correctly
measure the fractions of different components (e.g. aggregates) in a
multi-component mixture due to the Johnston-Ogston effect.</font></p>
<p><font size="2">John</font></p>
<p><font size="2">-----Original Message-----<br>
From: <a class="moz-txt-link-abbreviated" href="mailto:rasmb-bounces@rasmb.bbri.org">rasmb-bounces@rasmb.bbri.org</a> [</font><a moz-do-not-send="true"
href="mailto:rasmb-bounces@rasmb.bbri.org"><font size="2">mailto:rasmb-bounces@rasmb.bbri.org</font></a><font
size="2">] On Behalf Of Fumio Arisaka<br>
Sent: Saturday, April 05, 2008 4:20 AM<br>
To: <a class="moz-txt-link-abbreviated" href="mailto:rasmb@server1.bbri.org">rasmb@server1.bbri.org</a><br>
Subject: [RASMB] [Fwd: interference optics]<br>
<br>
Dear RASMBers,<br>
<br>
This concerns measurement of high concentrations of IgG with
interference optics. I have not much experience with IF, but have
started to use it for the necessity to measure high concentration
samples. Concentrations less than 5 mg/mL had no problem.<br>
Attached jpg file is to show the problem at 10mg/mL. Somehow, the
boudaries tend to go horizontally before reaching the plateau.<br>
<br>
I thought this was due to the misalignment of optics or something,
because the fringe pattern is not so good as I expect, but the
BeckmanCoulter person who came to fix it could not make it better.<br>
<br>
I would like to know what is the common highest concentration that you
could measure by IF. As I understand one could measure SE at higher
concentrations than 100 mg/mL, but when the boundary gets too steep in
SV measurement, the fringes get too much squeezed to count precisely.<br>
<br>
Any comments and advice are highly appreciated.<br>
<br>
Best wishes, Fumio<br>
<br>
<br>
<br>
<br>
</font></p>
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