[RASMB] Variability in association constants from sedimentation equilibrium data

[Toth IV, Ronald T]

Hello members!

We have a project where we are trying to extract association constants from sedimentation equilibrium data of a self-associating mAb. We are noticing a lot of variability in the association constants fit to between concentrations and even between replicates of the same concentration. My question is, is this expected? If not, what are some possible reasons it could be happening and what can I do to resolve it?

I’ll give more information in case it is relevant. We are using Beckman 6-sector cells and absorbance optics at 280 nm. Our initial guess as to rotor speed was a little high and gave sigma’s between 4 and 5. In some sectors there was a mismatch between sample and reference, but nothing in the buffer absorbs at 280 nm. In the fit we are using Heteroanalysis with a monomer-nmer model and constraining the molecular weight and stoichiometry.

We had a relatively new user load the cells, could cross contamination between reference and sample combined with a mismatched meniscus cause this?

Thanks in advance for your feedback!

- Ronald Toth

Post Doctoral Researcher
Macromolecule and Vaccine Stabilization Center
University of Kansas
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