DLS Vs SEC

James Fethiere James.Fethiere at BRI.NRC.CA
Wed Nov 22 18:24:32 EST 1995


Hello everybody,

Are there any strong believers in Dynamic Light Scattering for estimation
of the aggregation state of proteins?

My problem is the following:

I have a 18 kDa protein (on SDS-PAGE) that i concentrated to 
6.2 mg/ml.  This sample was diluted to 1.2 mg/ml in Tris-Hcl 20mM
pH 7.4, 0.1M NaCl and ran on the dynamic light scattering machine
after filtration through a 0.1 micron filter.  The results show an
estimated MW in the range of millions and a polydispersed sample.  Once
recovered it was filtered through a 0.02 micron filter;  the count rate
dropped by about 25%, and the estimated MW was in the 20kDa range with the
sample being monodispersed.  So, great!! my original sample is highly aggregated
i suppose, and that is why i do not get crystals.

But then, when i take the same sample at 6.2 mg/ml and run it on a SEC column
(Ultraspherogel SEC 2000 from Beckman) in the same Tris+NaCl buffer the sample elutes
at a monomeric MW, either when injected at 6.2 mg/ml or when diluted at 1.2 mg/ml.
No sign of aggregation whatsoever.

So which experiment should i believe, and do any of you have any suggestions
to confirm these results.

Thanks in advance for your advice.

James

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National Research Council of Canada       E-mail:James.Fethiere at BRI.NRC.ca
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