Separation of protein aggregates - Analytical Methods & Tools for Lyophilization

Separation of protein aggregates
18 April 2006
Laboratorytalk
A recent application by the NIAID Laboratory of Persistent Viral Diseases, Hamilton, Montana, demonstrates that Wyatt Technology's innovative Eclipse field flow fractionation (FFF) system achieves successful size-based separation of protein aggregates associated with neurodegenerative diseases. The findings of this analysis are of particular interest for molecular biology, nanotechnology and environmental analyses and are detailed in a new, free of charge application note from Wyatt, the world leader in absolute macromolecular characterization instrumentation and software. Neurodegenerative diseases such as Parkinson's, Alzheimer's and the transmissible spongiform encephalopathies (TSEs) are often marked by abnormal protein deposits in the brains of afflicted individuals.
Successful size-based separation of these aggregates, which can range from small protein oligomers to large amyloid fibrils, has been a challenge for traditional methods such as size exclusion chromatography (SEC), polyacrylamide gel electrophoresis (Page) and ultracentrifugation.
In both SEC and Page, large aggregates may be caught up in and/or subjected to shearing forces by the stationary phase, while ultracentrifugation techniques provide relatively limited resolution.
Wyatt's Eclipse overcomes the limitations of conventional column chromatography being able to separate both soluble and colloidal components over a wide size range.
Especially for proteins, the Eclipse is ideal since shearing forces and sample interactions with a stationary phase are completely absent.
The end result is an ultra-high resolution separation.
For this particular application, the Eclipse was used to separate a collection of prion protein aggregates, which were analyzed on line by the 18-angle Dawn EOS Mals (multi-angle light scattering), Optilab and WyattQELS (quasi-elastic light scattering) instruments to obtain weight-average molar mass (Mw), radii of gyration (rg), and hydrodynamic radii (rh).