Protein Aggregation (1-100 nm Soluble Aggregates)
Several methods are used to characterize soluble aggregates (Figure 1). Our center is equipped with the following instruments:
- Wyatt Dynapro DLS system
- Brookhaven DLS system
- Wyatt Dawn Heleos II MALS system
- Shimadzu® HPLC systems
- Beckman ProteomeLab XL-1 analytical ultracentrifuge
Dynamic Light Scattering (DLS)
The fluctuations of light intensity scattered from particles undergoing Brownian motion is the basis for DLS. To calculate the hydrodynamic diameter, an autocorrelation function yields a diffusion constant based on the Brownian motion of the particles. Our lab is equipped with a Wyatt Dynapro DLS system that performs DLS in sample volumes as small as 30 µl in 96- or 384-well plates as well as two Brookhaven DLS systems that can also measure the Zeta potential.
Static Light Scattering
Static light scattering (SLS) measures changes in the average intensity of scattered light, and if performed at different angles and concentrations, can determine changes in the radius of gyration of a protein molecule. We have available an SE-HPLC instrument in conjunction with UV absorbance, refractive index, and multi-angle light scattering (MALS) detectors to obtain absolute molecular weights of proteins and other macromolecules as well as soluble protein aggregates. In addition, we routinely use UV optical density spectroscopy to monitor the presence of aggregation through increases in optical density at 320-350 nm. It is also possible to obtain light scattering data during fluorescence experiments by monitoring the scattered light seen at the excitation wavelength using a second photomultiplier located at 180o to the fluorescence detector. Thus, information can be obtained about association/dissociation phenomena simultaneously with fluorescence emission data.
Analytical Size Exclusion High Performance Liquid Chromatography (SE-HPLC)
Two Shimadzu® HPLC systems are used exclusively for SE-HPLC. Small amounts of sub-micron aggregates (<1% of protein in solution) can be resolved and quantified using this method. These instruments are equipped with autosamplers to enable high-throughput analysis.
Analytical Ultracentrifugation (AUC)
While DLS determines the effective diameter of any particle that scatters light, analytical ultracentrifugation offers the benefit of limiting the measurement to particles that absorb light at a specific wavelength (e.g. 260 nm for DNA, 280 nm for proteins). Instead of calculating the effective hydrodynamic diameter, AUC measures the particle sedimentation coefficient. Our center has a Beckman ProteomeLab XL-1 analytical ultracentrifuge equipped with integrated Rayleigh Interference and Scanning UV/VISIBLE detection systems.