Home › Analytical Chem › Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)
Analytical Chem JoVE (Open Access) Citable · DOI

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)

DOI: 10.3791/59615-v
What you'll learn
  • ✓ Operate SEC-MALS instrument to determine absolute molecular weight of proteins
  • ✓ Prepare buffers, samples, and calibrate FPLC and MALS software systems
  • ✓ Analyze chromatography data to characterize protein complexes and oligomeric states
  • ✓ Interpret SEC-MALS results for native protein complexes and modified proteins
Protocol

This protocol describes the combination of size exclusion chromatography with multi-angle light scattering (SEC-MALS) for absolute characterization of proteins and complexes in solution. SEC-MALS determines the molecular weight and size of pure proteins, native oligomers, heterocomplexes and modified proteins such as glycoproteins.

Difficulty
intermediate
Total time
~3–4 hours per sample (including equilibration, chromatography run, and data analysis)
Biosafety
BSL-1

Steps

1
Prepare buffers, system, and load sample

Prepare appropriate buffers, equilibrate the SEC-MALS system, and load the protein sample onto the column for analysis.

▶ 00:57
2
Configure MALS and FPLC acquisition software

Set up detection parameters in the MALS software and configure the FPLC (fast protein liquid chromatography) control software for coupled operation.

▶ 03:42
3
Analyze SEC-MALS BSA chromatography data

Process raw chromatography and light scattering data from a BSA standard to demonstrate molecular weight determination and data interpretation.

▶ 05:49
4
Review SEC-MALS results and characterization

Examine final molecular weight, oligomeric state, and size outputs for protein samples and native complexes.

▶ 08:04
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