Ultrcentifugation: Basic Training, some slides out of Thermo Scientific education material, published with the permission of Richard Siccard.

1. Ultracentrifugation: Basic Training
Thermo Laboratory
Products

2. Applications
Smallest particles need strong g- forces for separation.
Superspeeds are not sufficient (only 50-100,000 xg max)
Ultras separate tiny particles with forces over 800,000 xg!!
Microultras separate small volumes - forces over 1,000,000 xg!!
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3. Applications
Important Terms in Ultracentrifugation
Abbreviation What it means
RPM Revolutions per Minute: The number of times a
rotor spins completely around in a single minute.
As RPM increases, RCF also increases.
RCF Relative Centrifugal Force: G-force developed in
a rotor while it spins. Many protocols use RCF
instead of RPM. As g-force increases, pelleting or
separation time decreases.
K K-Factor: Pelleting efficiency of a rotor. The
smaller the K-factor, the more efficient the rotor, or
the less time it takes to pellet particles.
S Sedimentation Coefficient: Depends on diameter,
shape, density. The larger the S value, the faster a
particle separates.
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4. Applications
• Smaller particles also
have very small S-
Density (g/ml)
values.
• High RCF needed to
pellet or separate
• Ultras required!
Sedimentation Coefficient (S)
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5. Applications
– As particle size/weight decreases, more
force needed to “pellet”
Differential
– Smaller particles remain in “supernatant”
Pelleting
– Fixed Angle rotors work best
• Ultra: T-8100, T-880, T-1270, etc.
• Microultra: S150-AT, S120-AT2, etc
– Example protocol - subcellular fractionation
• Spin 1: 1000xg to pellet nucleus
• Spin 2: 10000xg to pellet
mitochondria
• Spin 3: 100000xg to pellet
microsomes
• Spin 4: 900,000xg to isolate proteins
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6. Applications
Rate zonal density gradient centrifugation
Before After
• Good for particles with
Low similar densities, but
Density Sample
different masses
• Example particle: Proteins
• Example gradient material:
sucrose
High
Density
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7. Applications
Isopycnic (Equilibrium density gradient )
centrifugation
Before
• Separates particles according
After
to density
• Example particles:
– DNA, RNA, Plasmid DNA
– Viruses
– Organelles
• Example gradient: Cesium
Chloride
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8. Applications
Centrifugation Method Versus Rotor Type
Rotor Separation Considerations
Type
Swinging Longer separation time due to longer
bucket pathlength. Especially good
separation for Rate-Zonal separation.
Beware of Cesium Chloride quot;point
loadsquot; during isopycnic centrifugation.
Fixed Shorter separation time due to shorter
Angle pathlength. Excellent for simple
pelleting.
Vertical Shortest pathlength. Fastest
separation, especially for Isopycnic
separation. Pathlength may be too
short for rate-zonal separation. Pellet
smears along wall in simple pelleting.
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9. Applications
Type of Rate-zonal Rate-zonal
rotor Pelleting sedimentation flotation Isopycnic
Fixed- Excellent Limited Good VariableA
angle Ex. T-880,
S120-AT2
Vertical NS Good Good Excellent
Ex. Stepsaver,
S120-VT
Swinging Inefficient Good Excellent GoodB
Bucket Ex. TH-641,
S55-S
For Excellent rotor-applications,
a common ultra and microultra rotor are listed
NS = not suitable
AGood for macromolecules, poor for cells and organelles
BGood for cells and organelles, caution needed if used with CsCl
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10. Applications
Most Typical
Typical Appropriate Separation
Sample Applications Rotor Method
DNA and RNA Sequencing, Vertical and CsCl gradient,
gene therapy, Fixed angle 400,000xg;
cloning, gene Ethidium
expression Bromide staining
Viruses Vaccines, gene Swinging bucket Sucrose
therapy vector gradients,
100,000xg
Proteins Protein structure Fixed angle Rate-zonal
studies, separation,
Proteomics, 600,000xg
HDL/LDL studies
Cells and Cell function, Fixed angle Differential
organelles membrane pelleting. Low
biology, speeds for cells.
mitochondrial High speeds -
DNA smaller
organelles.
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11. Applications - Tube Selection
• Selection of the appropriate Properties of 4 popular tube plastics
ultracentrifuge tube Chemical
– Prevents sample leakage or Plastic type Clarity Resistance
loss Polypropylene (PP) Opaque Good
– Ensures chemical
compatibility Polyallomer (PA) Opaque Good
– Allows easy sample recovery
Polycarbonate (PC) Clear Poor
• Major factors in selection of a Polyethelyene Clear Poor
tube (plastic) material: Terephthalate (PET)*
– Clarity
– Chemical resistance PET is also in Polyclear™, Clearcrimp®,
– Sealing mechanism (if Ultraclear & other tubes
needed)
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12. Applications - Tube Selection
Tube type must be carefully matched with rotor type to prevent
sample loss and/or tube failure
Fixed Swinging
Tube type Puncture Angle Bucket Vertical
or slice Rotor Rotor Rotor
Thin wall Yes No Yes No
open top
Thick wall No Yes Yes No
open top
Thin wall Yes Yes Some Yes
sealed tube types
Oak Ridge No Yes No No
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13. Applications - Sample Collection
• Sample collection from tubes
(depends on application, gradient,
etc)
– Pour liquid from top if simple
pellet
– Draw liquid from top with pipette
– Use syringe to puncture tube &
draw out sample
– Cut tube with tube slicer
– puncture bottom & collect droplets
(fractions)
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14. Applications - Rotor Types
Review
• Fixed Angle – differential pelleting
• Vertical - isopycnic separation (density)
• Swinging Bucket – isopycnic or rate-zonal separation
(density or size)
• Others
–Zonal - rate zonal separation (size)
–Continuous-Flow – large volumes of sample
–Near Vertical – isopycnic separations (density)
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