There are two analytical ultracentrifuges: a Beckman Coulter XLI and a Beckman Coulter XLA. The XLI is equipped with both Rayleigh Interference and absorbance optics. This provides a complete analysis both at low concentrations (~0.1 mg/ml) and samples without chromophores. The XLA is equipped with only absorbance optics and is mainly used for protein/peptide analyses.
The information you can get from the analytical ultracentrifugation is dependent on the questions being asked. Routinely, sedimentation velocity experiments are employed. This type of experiment can be used to evaluate the stoichiometry and kinetics of interactions along with hydrodynamic and thermodynamic properties of the reacting systems. This technique can provide information on the sedimentation coefficient distributions, macromolecular shape distributions, equilibrium/rate constants and molecular weight distributions. Sedimentation equilibrium experiments can also be performed to determine kinetic constants for self associating systems. These experiments are conducted in the native buffer and do not rely on either immobilization or labeling of the samples.
The Biacore 3000 is built on the platform that uses the Surface Plasmon Resonance (SPR) phenomena, which can be used to monitor the refractive index of a given medium near the surface of a chip. The Biacore 3000 uses a label free system to determine the kinetic parameters (kon/koff) of biomolecular interactions. Evaluation of the kinetic parameters (kon/koff) of a protein-ligand system will require the immobilization of either protein or ligand to the surface of the chip. The binding of analytes flowing of over the surface of the chip can be uses to describe kon (association) and koff (disassociation). The chips are re-useable and have 4 binding surfaces which can be evaluated simultaneously. The Biacore Evaluation software can be used to derive the kinetic parameters for several models.
ITC (Isothermal Titration Calorimetry)
ITC is the only instrument capable of measuring the delta H (enthalpy) of interactions. The stoichiometry along with the strength of the interaction (Kd) can be ascertained from ITC experiments. This instrument is the gold standard for pharma companies to fine tune the drug interactions. The ITC is ideal for small molecule-protein based interactions but can be applied to any system e.g. Protein-DNA, RNA interactions. This system also does not require any specific labeling, immobilization or special buffers.
SEC-MALS (Size Exclusion Chromatography Multi-Angle Light Scattering)
SEC- MALS experiments can provide information on the absolute molecular weight of a system in solution without the need for standards, labeling or immobilization. The SEC-MALS also provides information about the hydrodynamic radius of the system and can be applied to a variety of different samples: proteins, DNA, RNA, polysaccarides, virus, polymers, nanoparticles etc. This technique coupled with AUC can provide a complete biophysical characterization of your particle’s size and distribution.
Cary Eclipse Fluorimeter
The Cary Eclipse fluorimeter can be used for a variety of fluorescent based assays. This instrument is also equipped with a manual polarizer for polarization assays and a 96-well plate adapter for larger sample analysis.
The BioAnalytical Lab in collaboration with the Purdue Proteomics Facility has access to the ABI 4800 mass spectrometer. The 4800 uses matrix assisted laser desorption/ionization (MALDI) to analyze biomolecules such as proteins, peptides and sugars. With each analytical ultracentrifugation analysis, a complementary mass spectrometric whole protein analysis is conducted. There are matrices which preserve the non-covalent interactions of proteins, ionic liquid matrices (ILM). We can also look at membrane proteins using the Ultra Thin Layer Method. The data gathered from these mass spectrometric analyses agree with the solution data from the analytical ultracentrifuge. Combined, both techniques provide a robust and complete evaluation of your samples.
Jia Ma, PhD
Bindley Bioscience Center
West Lafayette, IN 47907-1971