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 at the CMI
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. SEC-MALS is used to measure the weight-averaged molar mass of proteins (and other macromolecules) in solution, and 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 mass determination 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.
SEC-MALS System Components
- Wyatt Dawn Heleos II Multi-Angle Light Scattering (MALS) detector
- Wyatt Optilab T-rex Refractive Index Detector
- Wyatt In-line QELS detector for Dynamic Light Scattering
- Agilent 1260 Infinity Isocratic Liquid Chromatography System
- Agilent 1260 Infinity Autosampler
- inline solvent degasser
- variable wavelength UV detector
There is no fraction collector on this system, as it is intended for analytical and not preparative chromatography.
SEC-MALS Column Data
Generally, SEC-MALS data collection is done in ASTRA 7 with HPLC control.
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 Working with Custom Solvents, for solvents that cannot use PBS or water as the solvent model.
SEC-MALS Technology page from Wyatt Technologies.
- purified protein samples (must be filtered before loading)
- running buffer (1L)
- analytical SEC column (the CMI usually has 1-2 working SEC-MALS columns that users may borrow, if their samples are purified by SEC and in an appropriate buffer)
- 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.
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
- Scattering is proportional to mass
- 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.