An important part of characterizing a macromolecule is to determine its size and shape. Various instruments are available to measure the hydrodynamic properties of macromolecules including DLS, AUC, SEC coupled with MALLS and nanosight particle tracking (see quaternary structure and stability).
Our center is equipped with the following instruments:
- Beckman ProteomeLab XL-1 analytical ultracentrifuge equipped with integrated Rayleigh Interference and Scanning UV/VISIBLE detection systems
- Shimadzu® HPLC systems with a Wyatt Dawn Heleos II MALS detector
- Wyatt Dynapro DLS system
- Brookhaven DLS system
- NanoSight (LM10-HS)
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 or the molecular weight (including that of associating systems) using equilibrium sedimentation.
Size Exclusion Chromatography with MALS Detection
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. One of the HPLC systems is connected to a Wyatt Dawn Heleos II MALS detector. The Wyatt system is an 18-angle light scattering detector for the measurement of absolute molecular weight, size, and conformation of macromolecules in solution.
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. A variety of different data analysis methods are available, ranging from cumulant analysis to various deconvolution methods for multicomponent resolution.
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 and molecular weight 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.
Nanosight Particle Tracking
NanoSight (LM10-HS) visualizes, measures, and characterizes most nanoparticles (50 nm-2 µm) using a finely focused laser passed through a prism edged optical flat. Due to refraction, nanoparticles in liquid are observed using a microscope under 20X magnification. Particle size, concentration, zeta potential, and aggregation can all be analyzed using this method. NanoSight measures concentration and validates all data by recording short movies of particles moving under Brownian motion. The results are observed in real time by a CCD camera, while each particle is tracked by dedicated computer software. No information on refractive index or density is needed for particles.
He, F., Joshi, S.B., Moore, D.S., Shinogle, H.E. and Middaugh, C.R. (2009) Using spectroscopic and microscopic methods to probe the structural stability of human adenovirus type 4. Human Vaccines 6(2), 202-211
PMID 19946219: http://www.ncbi.nlm.nih.gov/pubmed?term=19946219