Size-Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS)
CMI SEC-MALS resources and user guide⬇︎
Size exclusion chromatography with multi-angle static light scattering (SEC-MALS) is used to accurately measure weight-averaged masses (Mw) of macromolecules in solution by measure the intensity of scattered light of a sample as it elutes from an SEC column. SEC-MALS can determine oligomeric state and sample polydispersity.
Size-exclusion chromatography (SEC) separates molecules based on hydrodynamic volume, but is dependent on similarity to a set of reference standards for accurate estimates of mass and fails for elongated or sticky proteins. Multi-Angle Light static Scattering (MALS) is used to measure light scattering intensity accurately, which is proportional to the weight-averaged mass in solution. Combining SEC, MALS and concentration detectors in an SEC-MALS experiment allows for more accurate mass measurements that SEC or MALS alone.
By using two concentration detectors (RI and UV), the molar mass and weight-fraction of a modifier can be determined. This can be used to measure protein and modifier masses of integral membrane proteins in detergent micelles and of glycosylated proteins.
The CMI has a SEC-MALS system with a Wyatt Dawn Heleos Multi-Angle Light Scattering (MALS, 18-angle) Detector, with in-line DLS detector and Optilab TrEX refractive index detector; Agilent 1260 Infinity Isocratic Liquid Chromatography System with autosampler (5-100 μl) and variable wavelength UV detector.
** There is no fraction collector on this system, as it is intended for analytical and not preparative chromatography.
Key Features
- Measure molar mass and oligomeric state for wide range of particle sizes
- proteins ~5MDa – 5kDa
- Deconvolute mass contribution from two components
- Protein and detergent micelle
- Protein and glycan
- Protein and DNA/RNA
- Determine hydrodynamic radius (Rh) from DLS data
- RMS radius (Rg) for large particles
SEC-MALS Resources
Generally, SEC-MALS data collection is done in ASTRA 7 with HPLC control.
CMI SEC-MALS Getting Started Guide
CMI SEC-MALS Guide to Protein Conjugate Analysis, guide to measuring dn/dc values and performing protein conjugate data analysis.
CMI SEC-MALS Guide to Custom Solvents, for solvents that cannot use PBS or water as the solvent model.
SEC-MALS Technology page from Wyatt Technologies.
SEC-MALS Supplies
- Sample filters, 0.02-0.2 μm filters (recommended)
- Autosampler vials (provided by the CMI)
- Analytical SEC Column:
- A range of analytical SEC columns can be used including silica and agarose columns
- silica columns (e.g. SEPAX SRT SEC-300, 7.8 mm x 300 mm)
- generally excellent performance
- low shedding
- maximum pH of 7.5
- agarose columns (e.g. Superdex 200 Increase 3.2 x 300mm)
- more shedding, requires longer equilibration times
- wide pH range
- The CMI usually has 2-3 working SEC-MALS columns that users may borrow, if their samples are purified by SEC and in an appropriate buffer (pH ≤ 7.5, salt > 100 mM). No guarantees on performance are offered.
- Make sure that the column is compatible with your sample. Some detergents and membrane proteins will require special columns designed for hydrophobic samples and buffers.
SEC-MALS Sample Preparation
Assay Buffers
- Running buffer should always be chosen to be compatible with both the SEC column and the protein sample.
- Recommended Buffer: 25 mM HEPES pH 7-7.5, 150 mM NaCl (filtered).
- Make sure you know the buffer compatibility of the SEC column you are using.
- Most silica columns will not tolerate pH above 7.5.
- Some buffer components (e.g. glycerol) will require customization of the solvent profile. Take note of the absolute refractive index of the solvent after equilibration and before data collection.
Samples
- The sample should be prepared in running buffer to minimize RI peak due to sample solvent.
- This is particularly important for samples that run near the solvent peak.
- It is good practice to run a size-exclusion chromatography purification prior to analytical SEC-MALS to buffer exchange and clear aggregates. This is a requirement if you are using a shared CMI column.
- Samples must be filtered or centrifuged prior to injection.
- Concentration should be accurately measured to assess column recovery.
- Know the concentration (in mg/ml) of your protein.
- Know the UV extinction coefficient (in ml/mg•cm) of your protein
- Protein aggregates can damage the column.
- Filter or centrifuge samples before use.
- Assess protein heterogeneity via dynamic light scattering (in the DynaPro plate reader).
- Purify protein samples with soluble aggregates by size-exclusion chromatography.
- Recommended protein concentration varies depending on protein mass:
- Scattering is proportional to mass
- Larger proteins require less sample than smaller proteins.
- Typical range 5 – 500 µg/injection
- BSA (67 KDa) 100 µl at 2 mg/ml always gives a good light scattering signal
- mg ~ 10/mass(KDa)
- Scattering is proportional to mass
- Sample volume:
- Maximum injection volume: 100 µl
- Minimum injection volume: 5 µl
- Glass autosampler vial with low volume insert has 10 µl dead volume.
- Fill vial with at least 110 µl for a 100 µl injection.