Dynamic and Multi-Angle Light Scattering

Dynamic and Multi-Angle Light Scattering

Please acknowledge the SIP core facility ( RRID: SCR_018986) in publications, on posters, or in talks if you use any instruments in the SIP core facility. Please include SIP's RRID (RRID: SCR_018986) and the grant numbers for instruments funded through instrumentation grants in your acknowledgements. This is a requirement from the funding agencies and is crucial for future funding. Find example text on theÌýAcknowledgement PageÌýor the individual instrument pages.

DLS and MALS in Biochemistry, Biophysics and Structural Biology

Macromolecules in solution scatter light from a culminated, monochromatic beam in all directions. ÌýThe scattering intensity and pattern are influenced by the size and shape of the macromolecules. The detected scattered light is analysed to yield information about the biomolecules and complexes in solution, including details on their size, shape, molecular weight, oligomeric states, homogeneity, and other key properties.

DLS – Dynamic Light Scattering

Dynamic light scattering (DLS) measures fluctuations in the intensity of scattered light, caused by the Brownian motion of particles. By analysing these fluctuations, DLS can determine the diffusion coefficients (DÏ„) of the scattering particles, which are directly related to their hydrodynamic radius. DLS is used to characterise the modality and size distributions of particles, including proteins, polymers, and nanoparticles in solution. This technique is particularly valuable for studying the stability and aggregation behaviour of biological macromolecules under various conditions, such as over time, or in response to changes in particle concentration, pH, or salt concentration, providing real-time insights into their dynamic processes. DLS is a useful tool for identifying optimal conditions for crystallisation, NMR, or Cryo-EM experiments, and for assessing stability in formulations.

MALS - Multi-Angle Light Scattering

Multi-Angle Light Scattering (MALS) measures the amount of light scattered by particles in solution relative to the angle of the incident light. For larger macromolecules and or for unfolded or strongly elongated proteins, the angular dependence observed in a multi-angle light scattering experiment can be used to calculate the radius of gyration (Rg), which reflects the mass distribution of a macromolecule around its centre of mass.

MALS used in conjunction with a concentration detector, can directly measure the absolute molar masses of macromolecules and complexes, independent of their structure and shape, across a wide range from 200 daltons to hundreds of millions of daltons. SEC-MALS, which integrates size-exclusion chromatography with an in-line MALS detector and a differential refractive index or UV/Vis detector can accurately determine the absolute molecular mass of proteins and complexes in complex samples, independent of their elution volume from the SEC column. Unlike conventional SEC, this method provides accurate molecular weight measurements even for sticky or elongated proteins that may elute too early or too late for their actual mass to be correctly determined by SEC alone, due to unusual interactions with the columns.

In composition-gradient multi-angle light scattering (CG-MALS), an automated composition-gradient preparative system is combined with an in-line MALS detector, a dRI detector and a UV detector to study interactions between biological macromolecules to determine Kd’s and stoichiometries of complex formation, without the need for labelling or immobilisation.

Methods and Instruments

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DLS data example showing fit and size distribution of BSA measured by DLS

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SEC-MALS Example showing results for a membrane protein in DDM micelles from conjugate analysis

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Key highlights of DLS in Biology, Biochemistry, and Biophysics

  • Small sample volume, non-destructive, and fast
  • Translational diffusion coefficient
  • Hydrodynamic radius
  • Size distribution
  • Polydispersity/Polymodality
  • Protein stability and aggregation as a function of temperature, concentration, or buffer conditions
  • Screening for optimal Cryo-EM, NMR, or crystallisation conditions
  • Screening for stability in formulations
  • And more...

Key Highlights of MALS in Biology, Biochemistry, and Biophysics

  • Absolute molar mass of macromolecules and complexes
  • Absolute molecular stoichiometry
  • Binding affinity, from pM to mM
  • Self- and hetero-association, including simultaneous self- and hetero-association
  • Second virial coefficient (A2)
  • Molar mass and stoichiometry of membrane protein-detergent complexes
  • Quantification of protein modifications (e.g., glycosylated or pegylated proteins)
  • ÌýNon-specific interactions between proteins at high concentrations
  • ÌýAnd more...

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DLS

Titan Pro DLS instrument

MALS

Wyatte Dawn Heleos II 18-angle static light scattering detector and a Wyatt Optilab rEX differential refractive index detector

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Calypso-II Continuous Gradient maker from Wyatt Technology

DLS Instrument and Accessories

Instrument

We have a Titan DynaPro instrument (formerly ProteinSolutions, now Wyatt Technology) for batch measurements. Data collection and evaluation are managed by the Dyna V6.3.4 software package.

Sample Holder

  • One single-cell holder for rectangular cuvettes.

DLS CuvettesÌý

  • Four shared 12 µl light scattering cuvettes with a special non-scattering coating.

Temperature ControlÌý

  • Peltier temperature control with an adjustable temperature range of 4 - 60°C.

MALS Instrument and Accessories

Instruments

We have two SEC-MALS systems, both equipped with individual MALS/dRI detectors that can also be used for batch measurements.

  • System Two: Wyatt Dawn EOS 18-angle light scattering detector combined in-line with a Wyatt Optilab DSP refractive index monitor, linked to a Shimadzu HPLC LC-20AD pump. An additional UV detector can be added to the system.
  • System One: Wyatt Dawn Heleos II, 18-angle light scattering detector in-line with a Wyatt Optilab rEX refractive index detector, linked to an ÄKTA purifier system for SEC and UV monitoring.

Accessories

  • Calypso: A composition gradient preparation system that can be combined with the Dawn Heleos II 18-angle light scattering detector, the Optilab rEX refractive index detector, and the ÄKTA purifier UV monitor for CG-MALS experiments.
  • QELS Detector: Integrated into the Dawn Heleos II for simultaneous DLS measurements.
  • Comet cell cleaner for the Dawn Heleos II.
  • Orbit Solvent Recycler integrated with the Dawn Heleos II set up.

Essential Information for Using SIP's DLS and MALS

Acknowledgements Protocols Training Scheduling Cost Location
Acknowledgements

Why?

Acknowledgements are essential for ensuring the continued success of the Shared Instruments Pool (SIP). They enable us to secure the necessary funding to sustain and expand the SIP, ensuring that our instruments are in optimal working condition and that the methods we offer are at the forefront of biochemical and biophysical research.

Please include SIP's RRID number (RRID: SCR_018986) in your acknowledgements. This allows funding organisations and potential grant reviewers to easily locate publications supported by SIP, helping to evaluate the impact of SIP on our research community.

If facility staff have provided substantial assistance, please consider acknowledging them. If they contributed significantly to the intellectual aspects or conducted important experiments, co-authorship may also be appropriate.

Example text:

We thank the Shared Instruments Pool (RRID: SCR_018986), Department of Biochemistry, University of Colorado Boulder for the use of the SEC-MALS (Wyatt Dawn Heleos II, Wyatt Optilab rEX , ÌýÄKTA purifier). We also thank [Name and title of the facility member providing significant help] for their invaluable assistance with data collection and evaluation.

We thank the Shared Instruments Pool (RRID: SCR_018986),Department of Biochemistry, University of Colorado Boulder for the use of the Titan Pro DLS. We also thank [Name and title of the facility member providing significant help] for their invaluable assistance with data collection and evaluation.

Please contact Dr Erbse to obtain detailed protocols and arrange an initial project consultation and personalised training sessions. Protocols are available as PDF files on the instrument computer, with printed copies stored alongside the instruments.

Interested users can contact Dr Erbse to discuss planned experiments and arrange for training. Training will involve the use of actual user samples alongside standards, enabling users to collect preliminary data with their samples of interest during the training and receive help from core staff right away if Ìýtroubleshooting or optimisation is needed. Users are welcome to request additional training or support sessions at any time. We are always happy to provide a refresher if it has been a while.

After your training is completed, you will be invited to join the SEC_MALS or DLS Google calendar.

Sign Up Rules:Ìý
Up to Friday the week before the planned experiment users can sign up for a maximum of 2 days. In the week of the experiment users can sign up for additional time if available.

  • Initial consultation is free. SIP staff are happy to assist with a short pilot experiment if it can be accommodated within SIP's resources.
  • Regular user groups are expected to buy into SIP with a monthly flat fee according to their SIP usage level. For detailed information, please contact Dr Annette Erbse.
  • Users are required to provide all consumables specific to their experiments.
  • Costs for necessary repairs, services, or replacement parts due to normal wear and tear will be shared among all user groups, based on the time used over the past two years. Please note that assuming the instrument is handled properly, such repairs or replacements are infrequent, and costs may arise after a user’s period of use has ended.
  • Users are responsible for covering the costs of repairs or replacement parts needed due to damage caused by carelessness or neglect.
  • The DLS is located on the in the basement of JSCBB in the C-Wing, room C1B60, on East Campus. Proxcard access is required at all times.
  • The SEC-MALS system One and the Calypso CG are located on the second floor of JSCBB, A Wing , room A355, on East Campus.
  • The SEC-MALS system Two is located on the third floor of JSCBB in the A-Wing, room A350, on East Campus.
  • Proxcard access is required at all times.