Hi,

What conditions did you use for the various analyses, for example regarding buffer/solvent composition and protein concentration?

Reversible self-association can be dependent on the ionic strength and/or concentration of the protein in solution.

Hope this helps!

Hi Eef,

For SEC and SEC-MALS, one injection do for both detectors, UV on HPLC and MALS. SEC gave heptamer based on protein standard curve, while MALS showed pentamer.

I would favor the result of SEC-MALS over SEC because SEC-MALS is not as dependent on the shape of the protein. Reversible association of dimers into larger oligomers may explain the difference between SEC-MALS and analytic ultracentrifugation if the concentration of protein is much higher in SEC-MALS than AUC.

Explanations for Your Observations:

SEC shows heptamer:

SEC separates proteins based on hydrodynamic radius, not actual molecular weight.

If your protein is elongated or asymmetric, it may elute earlier, mimicking a larger oligomer.

SEC can overestimate size if a smaller oligomer has a more extended shape.

SEC-MALS shows pentamer:

MALS measures absolute molecular mass independent of shape, assuming good signal and sample quality.

This is likely the most accurate representation of the true oligomeric state in solution, assuming no aggregation or degradation during the run.

A pentamer from MALS suggests your protein predominantly exists in that form under those conditions.

SV-AUC shows dimer:

SV-AUC is sensitive to population heterogeneity and may reveal smaller species if:

The protein dissociates during ultracentrifugation (e.g., weak oligomer)

You're analyzing at low concentration, shifting the equilibrium toward dimers

The buffer conditions destabilize higher-order assemblies

SV-AUC is also sensitive to shape, so frictional ratio (f/f₀) and diffusion coefficient modeling matter a lot.

Putting It Together:

Your protein may form a concentration- or buffer-dependent equilibrium between dimer, pentamer, and perhaps heptamer-like assemblies.

SEC overestimates oligomer size due to shape or compactness.

MALS gives the most reliable estimate for absolute MW in the context of SEC.

SV-AUC reveals that dimers are present, and possibly dominant under those (likely dilute) conditions.

What to Do Next:

Check sample concentration during SV-AUC. Run at multiple concentrations to see if there's a shift in sedimentation coefficient (suggesting dynamic equilibrium).

Compare buffer conditions between techniques.

Cross-validate with native PAGE or crosslinking if possible.

Consider cryo-EM or SAXS if structural details or shape effects are suspected.