Study of protein samples in solution using diffusion of light

Determination of the size and oligomeric state of a protein sample

Dynamic light scattering (DLS) is a non-destructive spectroscopic technique that allows to determine hydrodynamic radius and molar mass of molecules in solution. This method measures the scattering of particles subjected to Brownian motion and calculates the size distribution of the particles using the Stokes-Einstein relationship. The main applications of this technique are:

• Determination of hydrodynamic radius of a protein from which the apparent molecular weight can be deduced (if the protein can be considered as a globular particle).
• Knowledge of molecule folding state.
• Checking of sample homogeneity by determining the state of mono- or poly-dispersity.

The SEC-MALS technique allows to define the absolute molecular mass of the protein or complex analyzed regardless their apparent size and conformation. It includes exclusion chromatography (SEC) coupled online with a measurement of UV absorbance, laser light scattering (LS) and refractive index (RI). The proteins are separated by exclusion chromatography, the light scattered by the proteins is directly proportional to their molecular weight and their concentration.
The main applications of this technique are:

• Determination of molecular weight of proteins, protein/protein complexes, proteins/nucleic acids, nanodisks, etc.
• Determination of protein oligomerization state and stoichiometry of complexes.

Example of elution profile with the deduced molar mass of a protein of interest obtained with MALS Wyatt

Dynamic Light Scattering (DLS)

SEC- MALS