Applications of Ultracentrifugation in Purification and Characterization of Biomolecules
Content Type: Poster
Akash Bhattacharya,1 Ross VerHeul,2 Eric Von Seggern,1 Stephen Otts,2 Beckman Coulter, Inc., 1Loveland, CO & 2 Indianapolis, IN, USA
Ultracentrifuges spin samples with centrifugal forces typically spanning 100,000 – 1,000,000 x g. At these high forces, the constituent molecules in the sample separate based on their physical properties (e.g., size, mass, density, anisotropy). Accordingly, ultracentrifugation is commonly used to purify, as well as characterize, low-molecular weight polymers up to multi-megaDalton protein complexes and organelles.
- Particles are separated on the basis of their size and mass (sedimentation coefficient, S).
- Multiple pelleting steps may be used for iterative enrichment.
- Ideal for separating particle groups of very different sizes.
Density Gradient Ultracentrifugation (DGUC)
- Soluble particles are separated in a liquid column of varying density (density gradient)..
- In r ate zonal experiments, p articles migrate at varying rates, dictated by their S-values, and are time-dependent.
- Isopycnic separations are time-insensitive, where particles migrate to their apparent buoyant density in the gradient.
- Ideal for high-resolution separation of small materials with similar physical properties.
Example Workflow for DGUC Purification of Plasmid DNA
- Plasmid DNA may be extracted from bacteria using a variety of methods..
- The workflow below depicts a common alkaline lysis extraction and purification via a cesium chloride (CsCl) density gradient method with ethidium bromide.
- Newer density gradient materials (i.e., iodixanol/OptiPrep) and DNA-interacting probes (e.g., DAPI & GelGreen) may also be used in plasmid purification.