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

Introduction

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.

Preparative Ultracentrifugation

Preparative Ultracentrifugation 

Differential Ultracentrifugation

  1. Particles are separated on the basis of their size and mass (sedimentation coefficient, S).
  2. Multiple pelleting steps may be used for iterative enrichment.
  3. Ideal for separating particle groups of very different sizes.

 

Density Gradient Ultracentrifugation (DGUC) 

Density Gradient Ultracentrifugation (DGUC)

  1. Soluble particles are separated in a liquid column of varying density (density gradient)..
  2. In r ate zonal experiments, p articles migrate at varying rates, dictated by their S-values, and are time-dependent.
  3. Isopycnic separations are time-insensitive, where particles migrate to their apparent buoyant density in the gradient.
  4. Ideal for high-resolution separation of small materials with similar physical properties.

Example Workflow for DGUC Purification of Plasmid DNA

  1. Plasmid DNA may be extracted from bacteria using a variety of methods..
  2. The workflow below depicts a common alkaline lysis extraction and purification via a cesium chloride (CsCl) density gradient method with ethidium bromide.
  3. Newer density gradient materials (i.e., iodixanol/OptiPrep) and DNA-interacting probes (e.g., DAPI & GelGreen) may also be used in plasmid purification.

 

Example Workflow for DGUC Purification of Plasmid DNA

 

AUC History

figure10

Theodor Svedberg invents AUC,

Chemistry Nobel Prize 1926

figure11

Jean Perrin describes Sedimentation Equilibrium,

Physics Nobel Prize 1926

figure12

Ole Lamn describes the sedimentation and

diffusion of samples in a sector-cell, 1930s

figure13

Edward Pickel starts Spinco and builds the

first commercial AUC - the model E, 1947

figure14

Beckman acquires Spinco, 1954

figure15

Meselson & Stahi experiment, 1954

figure16

figure17

Schachman develops Rayleigh

interface detection, 1958


figure18

Van-Hoide Weischet graphical analysis

is developed, 1978

figure19

Beckman introduces the Proteomelab

XLA/XLI AUC instrument, 1990s

figure20

Demeler develops Ultrascan, 1998,

Shuck develops Sedfit, 2000

 

AUC Rotor & Optics

  • 4- and 8-hole rotors are rated to 60,000 and 50,000 rpm, respectively.
  • Interference and multiwavelength optics can be used in tandem.

figure6

figure3

Components of an AUC Cell

  • Two-sector centerpiece sandwiched between two quartz or sapphire windows & assembled into a cylindrical housing.
  • A screw ring is torqued to seal the cell.
  • A counterbalance is necessary.

figure4figure5

AUC Rotor & Optics

  • 4- and 8-hole rotors are rated to 60,000 and 50,000 rpm, respectively.
  • Interference and multiwavelength optics can be used in tandem.

figure6

Introduction to Sedimentation Velocity Data

  • Sample migation produces moving boundaries which contain sedimentation and diffusion information.

figure7

References: (1) By Thomasione, Public Domain, commons.wikimedia.org/w/index.php?curid=37665969.
(2) Ultrascan: www.ultrascan3.aucsolutions.com. (3) Sedfit: www.analyticalultracentrifugation.com.

AUC WORKFLOW CASE STUDY WITH INSULIN

Comparison of ProteomeLab vs Optima AUC

figure8

 

QUALITY CONTROL & CHARACTERIZATION USING AUC

Quantification of Aggregation and Degradation of Biological Samples and Elucidation of Molecular Shape

figure9

 

CENT-4959PST02.19

Contact Us