Light Scattering

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Size exclusion chromatography with multi-angle static light scattering (SEC-MALS) is used to measure masses of macromolecules.  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 accurately measure the intensity of scattered light, which is proportional to weight-averaged molar 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.  

Dynamic Light Scattering (DLS) measures the time-dependent fluctuations in scattered light. These fluctuations are directly related to rate of diffusion of the molecule through the solvent, and can be used to measure hydrodynamic radius (Rh) of the particles in solution. ​​​​​​

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.  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

DLS at the CMI

The CMI has Wyatt DynaPro Plate Reader III.  The DynaPro Plate Reader III measures dynamic light scattering (DLS) and Static Light Scattering (SLS) at high-throughput on many sample types in standard microwell plates. and can be used to rapidly assess protein quality in solution.  DLS measurements of hydrodynamic radius allow the monodispersity and aggregation state of a sample in solution to be measured in minutes and can be used to monitor protein quality during any (or all) stages of a protein purification project, or after storage.  DLS can also be used to size a variety of nanoparticles, including liposomes, micelles, bicelles, and nanodiscs and for aggegation analysis of chemical compounds. 

dynapro plate reader III

Wyatt DynaPro Plate Reader III Applications and Features

Applications

  • Rh and Mw of a wide range of particles (proteins, liposomes, nanoparticles and other macromolecules)
  • Monodispersity for rapid protein quality test
  • Protein stability from thermal denaturation
  • Aggregation analysis of samples, including chemical compounds
  • Multipeak Resolution
    • The peak resolution limit in dynamic light scattering is 5X in size.
    • Highly unlikely to resolve oligomers, such as dimers and trimers, from the monomer
      • Mixtures appear as a single broad peak
    • High-order oligomerization can be detected (>5-10mer)

Key Features

  •  Measure Rh from 0.5 nm to 1000 nm
  • Measure Mw from 1 to 1000 kDa
  • Sample volume from 4 μL to 150 μL
  • Sensitivity for size down to 12.5 μg/mL IgG
  • Measure in 96-1536 well microplates
  •  Temperature ramps from 4°C to 85°C

DLS Resources

CMI DynaPro DLS Getting Started Guide, guide to DynaPro Plate Reader III.

Tips for evaluating correlation function, adapted from DYNAMICS User’s Guide (M1406 Rev. F).

DLS Technology page from Wyatt Technologies.

DLS Supplies

0.02  μm filters for sample preparation, eg. Whatman Anatop 10, 0.02 μm, 10 mm inner diameter, Part Number 6809-1002

Clear-bottom Black Microplates:

number of wells 384 384 96
Manufacturer Aurora Microplates Greiner Bio-One Greiner Bio-One
Part Number ABM2‐10100A 781 892 655 892
base material 188 μm film glass glass
Minumum Volume 25 μl 50 μl 100 μl
Temperature Range 20-85 C 20-37 C 20-37 C
SLS Compatible yes yes yes
Recommended For high temperature/
thermal stability
Room Temp Room Temp

 

 

Assay Buffers

  • DLS is compatible with a range of buffers
  • Use filtered buffers
  • Measure scattering of buffer control
    • Detergents frequently have micelle size similar to that of proteins and scatter significantly

Samples

  • DLS is very sensitive to small amounts of aggregates.
    • Significant aggregates will make accurate size measurements difficult.
    • Samples should be filtered or centrifuged (6000g, 10-30 min) prior to transfer to the clear bottom plate to remove large aggregates and precipitates.
  • The minimum amount of sample required depends on the size of the molecule, as large molecules scatter more light than small molecules.
    • For most protein samples, concentrations of 0.2 mg/ml or higher are recommended.
    • As little as 0.125 mg/ml of lysozyme or 12.5 μg/ml of IgG can be detected.
  • Small molecule aggregation analysis should be done at working concentrations in and working solvents (e.g. 5% DMSO).
    • Solvent controls are essential. DMSO itself is prone to aggregation.
  • When working with membrane proteins, note that empty detergent micelles will not resolve from protein-filled micelles (except when empty micelle varies in size by 5-10X)
    • Avoid excess detergent by purifying membrane proteins by affinity and/or size-exclusion chromatography and avoid concentrating after purification

 

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.