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.
• The sample has to be filtered with 0.2 µm filter 1.5 mL reaction tube filters. Otherwise bring 65 µL of sample to be filtered on-site.Buffer
• Preparation of Running Buffer:

You will need: a 0.2 µm filter unit (single-use or with exchangeable membrane), two 500 mL bottles, and Milli-Q/ddH₂O.

1. Pre-rinse the filter
   1. Attach the filter unit to a 500 mL bottle.
   2. Filter 500 mL of Milli-Q/ddH₂O.
   3. Discard the filtered water and the bottle.
2. Rinse the buffer bottle
   1. Move the filter unit to a new bottle, which will hold your buffer.
   2. Filter 250 mL of Milli-Q/ddH₂O.
   3. Remove the filter unit, swirl the water to rinse the bottle walls, and discard the water.
   4. Repeat steps 2.2–2.3 once more, for two rinses in total.
3. Filter the buffer
   1. Place the filter unit back on the rinsed bottle.
   2. Filter your buffer.