[RASMB] Help needed

[Sophia Kenrick]

Dear Indrani,

As Tom mentioned, definitely look into the possibility of irreversible aggregation happening in parallel with your reversibly associating protein.  SEC combined with multi-angle light scattering is an ideal method for identifying the molecular weight of each species eluted from the column.  In the case of irreversible association, the aggregate peak should be completely separate from the monomer peak.  You can perform an SEC-MALS run on an aliquot of your protein after incubating for 1-15 hours to see if the fraction of irreversible aggregate grows with time.

In the case of reversible association, you should see an increase in Mw across a single peak as compared to the expected monomer molar mass for your protein.  For a reversibly associating protein, the main peak in an SEC chromatogram will shift to higher Mw as you inject a higher concentration on the column (or larger volume of the same concentration).  Depending on the kinetics of the reaction, you may be able to measure a higher molar mass at the very apex of the peak (where the concentration is highest) as compared to the leading or trailing edge where the concentration is lower.  However, often you get a constant, average Mw across the peak that changes as a function of the amount of protein that was loaded on the column.  You can get a semi-quantitative estimate of the KD from this type of experiment.

A more accurate quantification of oligomerization KD by light scattering would be performed in batch.  Composition gradient static light scattering (CG-SLS or CG-MALS) involves measuring the light scattering signal of different concentrations of an unfractionated macromolecular solution.  As complexes form, the weight average molar mass of the solution should increase.  The change in light scattering (Mw) as a function of composition can then be fit to an appropriate association model to yield affinity and stoichiometry.  Below is a link to a book chapter reviewing this technique, and the references listed in it may be useful to you.

http://www.intechopen.com/books/protein-interactions/characterization-of-protein-protein-interactions-via-static-and-dynamic-light-scattering

Best regards,
Sophia

Sophia Kenrick, Ph.D. | Application Development Engineer
Wyatt Technology Corporation
6300 Hollister Avenue | Santa Barbara, CA 93117-3253
Phone:  (805) 681-9009 x297 | Fax:  (805) 681-0123
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