The expertise of the members of Center for Drug Delivery and Nanomdicine (CDDN) in studying physico-chemical properties of nanomaterials and polymers has led to establishing the Nanomaterials Characterization Core Facility. The objective of the Core Facility is to provide investigators with state-of-the-art equipment, expertise and custom services for comprehensive study of polymers and nanomaterials. We are welcome all investigators to employ the Core capabilities in your research projects.
The COBRE Nebraska Center for Nanomedicine is supported by the National Institute of General Medical Science (NIGMS) grant 2P20 GM103480-07.
The Core Facility is equipped with:
Inductively Coupled Plasma Mass Spectrometer (ICP-MS)
PerkinElmer Nexion 300Q
Analytical Ultracentrifuge (AUC)
BeckmanCoulter Optima XL-I
Surface Plasmon Resonance (SPR)
Biacore 1000 upgrade and Biacore 3000
Multispectral Imaging System (MIS)
Kodak In-Vivo Multispectral Imaging System FX
Inductively Coupled Plasma Mass Spectrometer (ICP-MS) PerkinElmer Nexion 300Q
Offers best-in-class detection sensitivity by removing spectral interferences
High sample throughput
Determination of elements with atomic mass ranging from 7 to 250
Extreme sensitivity to achieve lower detection limits
Rapid multi-element quantitative analysis
Determination of metals concentration[TB1] in biological samples
AUC is the only technique with which you can determine accurately the native molecular weight of a protein and its assemblies with the polymers. This technique is also very powerful tool for characterization of supramolecular polymer complexes. For a typical sedimentation equilibrium experiment you only need 150 ml of a protein sample with an OD at 280 nm of 0.3. The obtained molecular weight is usually within 5% of the calculated value based on the protein sequence. AUC is applicable over a wide range of molecular weights from approx. 2.5 kDa up to 1.5 MDa. A typical experiment takes about 48 hours.
The stoichiometry of a protein complex can be calculated from the determined molecular mass. Depending on the quality of the data this determination can be very accurate. For instance, with high quality data it can be easily established whether the native conformation of a protein is a hexa- or a heptamer.
The assembly of a protein/macromolecule complex can be calculated from the determined molecular mass. It is even possible to follow the assembly when the different partners are added to the mixture one by one. Ligand binding can also be analyzed using sedimentation velocity methods if the ligand and acceptor differ greatly in their sedimentation coefficients. Alternatively, a thermodynamic analysis may be made using sedimentation equilibrium methods.
Conformation & Shape
Information about the shape and the conformation of a protein as well as the interaction between macromolecules can be obtained from the sedimentation and diffusion coefficients obtained from a sedimentation velocity experiment. Sedimentation coefficients are particular useful for monitoring changes in conformation of a protein. The resulting model for the overall shape of the protein or protein complex can be compared with images obtained by electron microscopy to assess how applicable those images are to the behavior of these particles in solution.
Unlike other methods for the study of binding processes, the sedimentation equilibrium method is particularly sensitive for the study of relatively weak associations with association constants (Ka) in the order of 10-100 M-1. However, binding processes with Ka values significantly greater than 107 M-1 can be also studied.
In a sedimentation velocity experiment, an initially uniform solution is placed in the cell and a high speed is used to cause rapid sedimentation of solute towards the cell bottom. This produces a depletion of solute near the meniscus and the formation of a sharp boundary between the depleted region and the uniform concentration of sedimenting solute (the plateau). The rate of movement of this boundary can be measured. This leads to the determination of the sedimentation coefficients, which depends directly on the mass of the particles and inversely on the frictional coefficients, which is in turn a measure of effective size.
Surface Plasmon Resonance (SPR) Biacore 1000 upgrade and Biacore 3000
The Nanomaterials Characterization Core Facility at UNMC currently has two Biacores: the Biacore 1000 upgrade and the Biacore 3000. BIAsimulation software version 2.1 and BIAevaluation software version 4.1 are available. The Biacore is an optical biosensor that uses surface plasmon resonance (SPR) for real-time monitoring of macromolecular interactions without the use of labels. The technology is for use in drug discovery, antibody screening, ligand fishing and therapeutics. Technological advantages include minimal sample consumption, recovery of surface bound samples for MS analysis, multi-sample analysis and measurement of weak binding events; furthermore, Biacore 3000 samples can include small molecules, crude extracts, lipid vesicles, DNA, RNA, viruses, bacteria or eukaryotic cells. Applications inlcude:
Kodak In-Vivo Multispectral Imaging System FX
The Kodak In-Vivo Multispectral Imaging System FX combines multispectral imaging with high-resolution x-ray imaging. The fully automated system’s powerful multispectral analysis software identifies and separates multiple fluorchromes which are spatially co-registered on the image. In addition, the system is capable of detecting luminescence and radioisotopic signals.
To submit samples for analysis, discuss experiment design or get additional information please contact Core Facility Manager Chantey Morris, PhD (firstname.lastname@example.org).