Equipment available: Viscotek 802
Equipment location: New Biochemistry 00-059
Equipment coordinator: Dr David Staunton
Equipment charge: £10/day
Sample requirements: 20ul of 0.1mg/ml protein
Dynamic Light Scattering
Dynamic light scattering (DLS) or quasi elastic light scattering (QELS) observes the intensity fluctuations with time of scattered light from a sample in solution. These fluctuations are related to the Brownian motion of the sample molecules thus their diffusion coefficient and thus their hydrodynamic radius Rh. Rh can be measured down to 1nm.
The intensity fluctuations with time are analysed by an autocorrelation function. The resultant autocorrelation plot consists of exponential time constants for all the particle components in the sample and can also give information on the heterogeneity of the sample. These distributions are analysed by regularization to give the Rh of the species present but this is cannot resolve species less than a factor of 5 different in Rh. Therefore DLS cannot be used to follow dimerization except under circumstances where the complex Rh is five times or more greater than the monomer Rh.
Sample characterisation. The small sample volumes makes DLS excellent for characterising the solution behaviour of proteins early in the purification process and can be used to screen samples for aggregation.
Protein stability. Rh can be followed with increasing temperature to determine the Tm as the protein unfolds and the Rh increases or aggregates.
Complex formation. For large complexes composed of multiple proteins the formation can be followed by DLS with increasing protein concentration.
Shape determination. The Rh can be approximated to half the longest axis of the protein or complex and so depends on the shape of the molecule. For large proteins (MW>200kDa) the compactness of the molecule can be characterised by comparing the Rh from DLS and the rms radius Rg from SEC MALS. For example a sphere has a Rg / Rh of 0.77 and an extended form has a value of 1.4.
The Viskotek 802 uses a 660nm laser with a micro fluorescence cuvette of sample volume 20 µl. The scattered light at 90° is measured by an Avalanche Photo Diode (APD) at a rate of one photon per µsec. Since the Brownian motion is sensitive to temperature and viscosity of the sample buffer, the temperature of the cuvette holder is set and the viscosity value selected from a library of common buffers or inputted directly.
Ten 10 second measurements are taken with the automatic data filter. If a spike appears in the data (from a large particle passing through the beam) that run will be terminated. Successful runs can be processed en masse to give a distribution of particle sizes.
Due to viscosity issues protein concentrations below 10mg/ml are used and only the solution viscosity considered.
The sample should be filtered if possible before analysis and we usually use 0.02 µm Anotop filters for this.
Readings should be taken from a dilution range where a consistent Rh is obtained for a series of concentrations.
Scattering is proportional to Rh6 so only a few large particles can dominate the signal from a small, monomeric protein.
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