[RASMB] Data analysis with Sedfit: Smin setting affect the sedimentation profile and how we should deal with it?

[Yi-Ming_Li at hgsi.com]

Hi Everyone:

I am using analytical ultracentrifugation for quantitative detection of
aggregates in purified antibodies. Our experiments showed that the antibody
is not self-associated in the buffer system used for velocity
sedimentation. The molecular weight of the antibody is about 150 kd. The
sedimentation conditions are as follows:

Rotor speed: 40000 rpm
Temperature: 20C
Running time: 6 hours

When I used Sedfit to analyze the AUC data, I found that S(min) setting
affect the sedimentation profile quite significantly. I first set the
S(min) to 0.2 s and S(max) to 20 s. With this setting, I got a profile with
96.8% of monomer, 1.8% of dimer and 1.4% of species larger than dimer as
showed in panel A in the figure. Since no peak was detected between 0.2-2 s
(although a spike of the base line at the Smin end was found), I reloaded
the data, and set the sedimentation coefficient range to 2-20 s for data
process. This time I got a profile with 98.9% monomer, 1.1% dimer, and no
species larger than dimer as showed in panel B in the figure. Moreover, the
sedimentation coefficient of the dimer is different in the two profile (8.9
s and 9.7 s).

It is important for us to know: 1) Is the way I set the S range right? 2)
Are the species larger than dimer found in panel A true species or just
artificial? 3) why the Smin setting affect the profile so much?

I would appreciate very much for any commend or discussion.

Thanks!

Yi-Ming

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