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Dynamic Light Scattering (DLS)

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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. ​​​​​​

DLS Instrumentation

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