#  Dynamic Light Scattering (DLS) 

 



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[**CMI DLS resources and user guide**](/node/1655856#dls-resources)**⬇︎**

[Dynamic Light Scattering (DLS)](https://www.wyatt.com/solutions/techniques/dynamic-light-scattering-nanoparticle-size.html) 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 allows 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.

 ![DLS example](/sites/g/files/omnuum4016/files/cmi/files/dls_theory_example.png)

 

The CMI has [Wyatt DynaPro Plate Reader III](https://www.wyatt.com/products/instruments/dynapro-dynamic-light-scattering-plate-reader.html). 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.

   ![dynapro plate reader III](/sites/g/files/omnuum4016/files/styles/hwp_1_1__360x360_scale/public/cmi/files/dynapro-plate-reader-iii-product.jpg?itok=L91_yQPA) 

 

**Applications**

- Rh 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**

- DLS to measure Rh from 0.5 nm to 1000 nm
- Bulk SLS to measure Mw from 1 to 1000 kDa (most meaningful for highly monodisperse samples)
- Standard sample volume from 25 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 DyanPro DLS Supplies DLS Sample Preparation 

## DLS Resources

[CMI DynaPro DLS Getting Started Guide](/sites/g/files/omnuum4016/files/2025-03/CMI%20Dynapro%20DLS%20Getting%20Started%20Guide.pdf "CMI Dynapro DLS Getting Started Guide"), guide to DynaPro Plate Reader III.

[CMI Dynapro SLS Getting Started Guide](/sites/g/files/omnuum4016/files/2025-03/CMI%20Dynapro%20SLS%20Getting%20Started%20Guide.pdf "CMI Dynapro SLS Getting Started Guide"), guide to DLS with static light scattering measurements.

[Tips for evaluating correlation function](/sites/g/files/omnuum4016/files/2025-03/DLS_Evaluation_Criteria.pdf "DLS_Evaluation_Criteria"), adapted from DYNAMICS User’s Guide (M1406 Rev. F).

[DLS Technology](https://www.wyatt.com/solutions/techniques/dynamic-light-scattering-nanoparticle-size.html) page from Wyatt Technologies.

 

 

 

## DyanPro DLS Supplies

- Filter for sample preparation: 0.2 μm minimum, 0.02 μm recommended (e.g. Whatman Anatop 10, 0.02 μm, 10 mm inner diameter, Part Number 6809-1002)
- Wyatt-validated DLS compatible Clear-bottom Black Microplate 
    - one 384-well plate provided to each user with training
    - additional plates available for purchase from the CMI at cost, for academic users

Sort**number of wells**

384

384

**Manufacturer**

Aurora Microplates

Greiner Bio-One

**Part Number**

ABM2‐10100A

781 892

**base material**

188 μm film

glass

**Minumum Volume**

25 μl

50 μl

**Temperature Range**

20-85 C

20-37 C

**SLS Compatible**

yes

yes

**Recommended For**

high temperature/  
thermal stability

Room Temp





 

 

 

## DLS Sample Preparation

**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.
    - ~ 10/mass(KDa)
    - 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

 

 

 

 

 

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