Faculté des sciences Faculté des sciences

SEC-MALS

Size-Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS)

SEC-MALS couples a size-exclusion chromatography to multi-angle light scattering, a refractive-index (concentration) detector, and UV absorption. The combination yields the absolute molar mass of each eluting species — independent of size-exclusion chromatography calibration or molecular shape — due to scattering intensity being proportional to molar mass times concentration.

Experiment Examples

  • Determining whether a purified protein is a monomer, dimer, or higher oligomer in solution, with an exact mass determination.
  • Measuring the absolute mass of protein/protein or protein/nucleic acids complexes to determine stoichiometry.
  • Quantifying the percentage of soluble aggregate (high-molecular-weight species).
  • Measuring the protein and glycan mass fractions of a glycoprotein separately.
  • Determining the mass of the protein component of a detergent-solubilized membrane protein, excluding bound detergent.

Sample Requirements

  • Standard molar-mass determination: 50 – 200 µg per injection; concentration 1 – 5 mg/mL in 60 µL.
  • Complex formation analysis: Sample containing components separately and in complex. Concentrations and volumes as above; amounts comparable for each sample. Ensure the sample is in the final running buffer (same buffer used to equilibrate the column).
  • Conjugate analysis (glycoproteins, PEGylated proteins, detergent-bound membrane proteins): Requires slightly more material.
  • Membrane proteins: Similar requirements as above. Use detergent beloe its CMC in the running buffer;
  • Buffer constraints: Filtered (0.05 µm), degassed, particulate-free running buffer is essential. Salt ≥150 mM is recommended to reduce non-specific column interactions.

Practicalities

  • Running buffer should be made fresh, filtered, and provided a day before. The system needs to be equilibrated overnight, at minimum, before measurements.
  • For filtering, you need to wash your 0.2 µm filter. Then clean your bottle with filtered water, and finally filter your fresh buffer into this bottle.
  • The sample has to be filtered with 1.5 mL reaction tube filters.