Dynamic Light Scattering (DLS), sometimes referred to as photon-correlation spectroscopy (PCS) or Quasi-Elastic Light Scattering (QELS), is a well established technique that measures particle size distribution in the nanometre region. Particles in solution are constantly bombarded by solvent molecules, inducing the particle’s uncorrelated random motion, or Brownian motion. When the particle solution is illuminated by a laser, scattered light from the randomly moving particles adds both constructively and destructively leading to time-dependent intensity fluctuations. By using a fast photon counter, the time-dependent intensity fluctuations, which are directly related to the rate of diffusion of the particle through the solvent, can be measured. Thus, analyzing the time-dependent intensity fluctuations can determine the hydrodynamic radius of a particle using the Stokes-Einstein relation.
Dynamic Light Scattering (DLS), is a well established technique to measure the hydrodynamic radius of particles in solution. The MS800 DLS instrument from Protein Solutions Inc. (now incorporated into Wyatt Technologies) can measure particles with hydrodynamic radii from 0.5 to 50 nm. DLS measurements are non-destructive, and measurements can be med very quickly with samples that do not exceed a radius of 50nm. This high sensitivity instrument can make measurements on as little as 100 μg mL-1 of a 14 kDa protein.
Typical particles measured by DLS include:
|Size Range||0.5 to 50 nm hydrodynamic radius|
|Laser Diode||830 nm (power adjustable)|
|Temperature Range||4 to 60 °C|
|Sensitivity||0.1 mg mL-1 for 14 kDa protein at 20 °C|
|Cuvette||Hellma 105.252-QS (b=1.5 mm, Z=15mm, V=12 μL)|
The sample concentration needed for DLS experiments will be dependent on the MW of the sample. The DLS is a very sensitive technique which requires sample concentrations as little as 100 μg mL-1 for a 14 kDa protein at 20 °C. Since larger proteins have a higher light scattering intensity, they may require less concentration. The Dynamics software used on the instrument can be used to calculate minimum sample concentrations. It is generally recommended that a minimum 1 mg mL-1 concentration is used for routine protein work and also a minimum of 50 μL of sample should be available. The sample is recoverable.
Sample must be contaminant free. Aggregates and dust must be removed by filtration (Anodisk filter 0.02 μm and 0.1 μm) or by centrifugation at 5000G for 30min. Sample cuvette should be thoroughly cleaned before any experiment.