DLS Vs SEC
James.Fethiere at BRI.NRC.CA
Wed Nov 22 18:24:32 EST 1995
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.
National Research Council of Canada E-mail:James.Fethiere at BRI.NRC.ca
Biotechnology Research Institute office: (514) 496-6173
Macromolecular Crystallography group lab : (514) 496-6376
6100 Royalmount Avenue fax : (514) 496-5143
Montreal, Quebec H4P 2R2
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