MULTI-ANGLE LIGHT SCATTERING (SEC-MALS)

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Multi-angle light scattering (SEC-MALS)

Size exclusion chromatography with multi-angle light scattering (SEC-MALS) is used to measure macromolecular mass (MW) in solution. SEC-MALS combines size-exclusion chromatography (SEC), multi-angle light scattering (MALS) and two concentration detectors based on the differential refractive index (dRI) and UV absorbance respectively.
Unlike SEC alone, the estimation of MW by SEC-MALS is absolute and is not based on the elution volume of macromolecules from an SEC column and its comparison with a calibration curve prepared with standard proteins.
In SEC-MALS, size exclusion chromatography is only used to separate the different entities/macromolecules present in the sample. The MW for a particular macromolecule is calculated from the amount of light scattered by the corresponding fraction relative to the laser intensity, the concentration of the macromolecule determined by the UV or dRI detector, and the difference between the refractive index of the analyte and the buffer.
SEC-MALS is not limited to proteins that behave in the SEC column similarly to standard proteins. It can be used for intrinsically disordered proteins, non-spherical or linear oligomeric assemblies, glycosylated proteins, detergent-solubilized membrane proteins, or proteins undergoing non-ideal column interactions.
SEC-MALS is applied to determine the MW of macromolecules separable by SEC, analyze their purity, establish their oligomeric state, examine the formation of macromolecular complexes, determine their stoichiometry, or assess the mass contribution from two components, such as protein and detergent micelle or protein and glycan.

Highlights

Instrument

Required Supplies

Manuals

SEC-MALS getting started guide

ASTRA software

Dedicated to running and analyzing MST experiments. It guides the user step-by-step through planning, setup and execution of experiments and helps to evaluate and analyze measured data.

DYNAMICS software

Used for analysis and evaluation of MST data.

References

Duhr S, Braun D. Why molecules move along a temperature gradient. Proc Natl Acad Sci U S A. 2006; 103:19678-19682. https://doi.org/10.1073/pnas.060387310
Jerabek-Willemsen M, Wienken CJ, Braun D, Baaske P, Duhr S. Molecular interaction studies using microscale thermophoresis. Assay Drug Dev Technol. 2011; 9:342-353. https://doi.org/10.1089/adt.2011.0380
Jerabek-Willemsen M , André T , Wanner R , Marie Roth H , Duhr S, Baaske P , Breitsprecher D. MicroScale Thermophoresis: Interaction analysis and beyond. Journal of Molecular Structure 2014; 1077:101-113 http://dx.doi.org/10.1016/j.molstruc.2014.03.009
Langer A, Bartoschik T, Cehlar O, Duhr S, Baaske P, Streicher W. A New Spectral Shift-Based Method to Characterize Molecular Interactions. Assay Drug Dev Technol. 2022; 20:83-94. https://doi.org/10.1089/adt.2021.133 Erratum in: Assay Drug Dev Technol. 2022; 20:136. https://doi.org/10.1089/adt.2021.133.correx
Seidel SA, Wienken CJ, Geissler S, Jerabek-Willemsen M, Duhr S, Reiter A, Trauner D, Braun D, Baaske P. Label-free microscale thermophoresis discriminates sites and affinity of protein-ligand binding. Angew Chem Int Ed Engl. 2012;51:10656-10659. https://doi.org/10.1002/anie.201204268
Seidel SA, Dijkman PM, Lea WA, van den Bogaart G, Jerabek-Willemsen M, Lazic A, Joseph JS, Srinivasan P, Baaske P, Simeonov A, Katritch I, Melo FA, Ladbury JE, Schreiber G, Watts A, Braun D, Duhr S. Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions. Methods. 2013;59:301-15. https://doi.org/10.1016/j.ymeth.2012.12.005
Wienken CJ, Baaske P, Rothbauer U, Braun D, Duhr S. Protein-binding assays in biological liquids using microscale thermophoresis. Nat Commun. 2010;1:100.