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Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering SEC-MALS
Published on: June 20, 2019
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Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)
Daniel Some1, Hadar Amartely2, Ayala Tsadok3
1Wyatt Technology Corporation; dsome@wyatt.com.
Journal of Visualized Experiments : Jove
|July 9, 2019
Summary
Size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) accurately determines protein molecular weight and size. This absolute method overcomes limitations of relative techniques, enabling robust characterization of protein solutions.
Area of Science:
- Biophysical chemistry
- Analytical biochemistry
- Protein characterization
Background:
- Analytical size-exclusion chromatography (SEC) is a relative technique for estimating protein molecular weight based on elution volume.
- SEC's accuracy is compromised by non-globular proteins or non-ideal column interactions, invalidating standard calibration curves.
- Multi-angle light scattering (MALS) provides an absolute molecular weight determination independent of protein shape or column interactions.
Purpose of the Study:
- To present a protocol for SEC-MALS analysis of protein molecular weight and size.
- To highlight SEC-MALS as a reliable method for characterizing protein monomers, aggregates, and complexes in solution.
- To establish SEC-MALS as a critical tool for confirming biophysical properties of proteins for research and biotechnology.
Main Methods:
- Coupling size-exclusion chromatography (SEC) for separation with multi-angle light scattering (MALS) for absolute detection.
- Utilizing MALS measurements at each elution volume to assess peak homogeneity and molecular weight distribution.
- Applying the SEC-MALS technique to analyze pure protein monomers and aggregates.
Main Results:
- SEC-MALS provides accurate, absolute molecular weight determination for proteins in solution.
- The technique effectively characterizes protein solutions containing monomers, native oligomers, aggregates, and heterocomplexes.
- SEC-MALS can distinguish between homogeneous and heterogeneous eluting peaks and identify dynamic equilibria.
Conclusions:
- SEC-MALS is a versatile and reliable method for comprehensive protein characterization in solution.
- This technique is essential for analyzing modified proteins like glycoproteins, lipoproteins, and membrane proteins.
- The presented protocol serves as a foundation for advanced SEC-MALS applications in protein science.
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