[RASMB] Why loading concentration affect main peak percentage so much?

[Yi-Ming_Li at hgsi.com]

Hi everyone:

I am trying to find a good buffer system for investigating the size
distribution of a protein sample (isoelectrical point 6.2, UV extinction
coefficient 0.62). In order to see whether self-association exists in the
buffer system, I diluted the samples to 2-, 1-, and 0.3 mg/ml and loaded
into 3 cells (3 mm certerpiece was used for 2 mg/ml loading) for
sedimentation velocity run. The C(S) profile of all loadings showed well
separated monomer, dimer and perhaps trimer peaks. But the percentages of
the main peak are very different between the 0.3 mg/ml and other loading
concentration (95.7% at 0.3 mg/ml, 98.9% at 1 mg/ml , 98.0 at 2mg/ml).
Moreover, the change of weight average sedimentation coefficient with
loading concentrations is complicated (4.94 s at 0.3 mg/ml, 4.80 s at 1
mg/ml and 4.84 s at 2 mg/ml). It seems to me that in 0.3-1 mg/ml
concentration range, self-association is negligible since the Sw decreases
from 4.94 s to 4.80 s when loading concentration increases from 0.3 mg/ml
to 1 mg/ml. But at high concentration, it seems that weak self-association
exists, since the Sw has little change or even increase a bit when loading
concentration increases from 1 mg/ml to 2 mg/ml. Our size exclusion
chromatography showed that the main peak percentage of the sample is over
99%. Therefore the result from 1 mg/ml loading AUC data is more consistent
with the size exclusion chromatography. I would like to know why the main
peak percentage is so different at 0.3 mg/ml loading. Is it my buffer
system (10 mM MES, 500 mM NaCl, 0.01% Tween 80, pH 5.5) is not good for
size distribution analysis? The attached is the C(S) profile.

Thanks for help!

Yi-Ming

(See attached file: Comparison of C(S) at two loading concentrations.ppt)
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