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

SEC-MALS Instrumentation

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.

 

 

Wyatt sec-mals system

SEC-MALS System Components

MALS

  • 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

Chromatography

  • 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 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 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)

 

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
  • 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.