ultrcentifugation: basic training
Post on 29-May-2015
5.194 Views
Preview:
DESCRIPTION
TRANSCRIPT
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!!
3
Applications
Abbreviation What it means
RPM Revolutions per Minute: The number of times arotor spins completely around in a single minute.As RPM increases, RCF also increases.
RCF Relative Centrifugal Force: G-force developed ina rotor while it spins. Many protocols use RCFinstead of RPM. As g-force increases, pelleting orseparation time decreases.
K K-Factor: Pelleting efficiency of a rotor. Thesmaller the K-factor, the more efficient the rotor, orthe less time it takes to pellet particles.
S Sedimentation Coefficient: Depends on diameter,shape, density. The larger the S value, the faster aparticle separates.
Important Terms in Ultracentrifugation
4
Applications
• Smaller particles also have very small S-values.
• High RCF needed to pellet or separate
• Ultras required!
Sedimentation Coefficient (S)
Den
sit
y (
g/m
l)
5
Applications
– As particle size/weight decreases, more force needed to “pellet”
– Smaller particles remain in “supernatant”
– 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
Differential
Pelleting
6
Applications
• Good for particles with similar densities, but different masses
• Example particle: Proteins
• Example gradient material: sucrose
Rate zonal density gradient centrifugation
Before After
Low
Density
High
Density
Sample
7
Applications
• Separates particles according to density
• Example particles:
– DNA, RNA, Plasmid DNA
– Viruses
– Organelles
• Example gradient: Cesium Chloride
Isopycnic (Equilibrium density gradient )
centrifugation
Before After
8
Applications
Centrifugation Method Versus Rotor Type
RotorType
Separation Considerations
Swingingbucket
Longer separation time due to longerpathlength. Especially good
separation for Rate-Zonal separation.Beware of Cesium Chloride "point
loads" during isopycnic centrifugation.FixedAngle
Shorter separation time due to shorterpathlength. Excellent for simple
pelleting.
Vertical Shortest pathlength. Fastestseparation, especially for Isopycnicseparation. Pathlength may be too
short for rate-zonal separation. Pelletsmears along wall in simple pelleting.
9
Applications
Type of
rotor Pelleting
Rate-zonal
sedimentation
Rate-zonal
flotation Isopycnic
Fixed-
angle
Excellent
Ex. T-880,S120-AT2
Limited Good VariableA
Vertical NS Good Good Excellent
Ex. Stepsaver,S120-VT
Swinging
Bucket
Inefficient Good Excellent
Ex. TH-641,S55-S
GoodB
For Excellent rotor-applications,
a common ultra and microultra rotor are listed
NS = not suitableAGood for macromolecules, poor for cells and organelles
BGood for cells and organelles, caution needed if used with CsCl
10
Applications
SampleTypicalApplications
MostAppropriateRotor
TypicalSeparationMethod
DNA and RNA Sequencing,gene therapy,cloning, geneexpression
Vertical andFixed angle
CsCl gradient,400,000xg;EthidiumBromide staining
Viruses Vaccines, genetherapy vector
Swinging bucket Sucrosegradients,100,000xg
Proteins Protein structurestudies,Proteomics,HDL/LDL studies
Fixed angle Rate-zonalseparation,600,000xg
Cells andorganelles
Cell function,membranebiology,mitochondrialDNA
Fixed angle Differentialpelleting. Lowspeeds for cells.High speeds -smallerorganelles.
11
Applications - Tube Selection
Plastic type ClarityChemical
Resistance
Polypropylene (PP) Opaque Good
Polyallomer (PA) Opaque Good
Polycarbonate (PC) Clear Poor
PolyethelyeneTerephthalate (PET)*
Clear Poor
• Selection of the appropriate ultracentrifuge tube
– Prevents sample leakage or loss
– Ensures chemical compatibility
– Allows easy sample recovery
• Major factors in selection of a tube (plastic) material:
– Clarity
– Chemical resistance
– Sealing mechanism (if needed)
Properties of 4 popular tube plastics
PET is also in Polyclear™, Clearcrimp®,
Ultraclear & other tubes
12
Applications - Tube Selection
Tube type Punctureor slice
FixedAngleRotor
SwingingBucket Rotor
VerticalRotor
Thin wallopen top
Yes No Yes No
Thick wallopen top
No Yes Yes No
Thin wallsealed
Yes Yes Sometube types
Yes
Oak Ridge No Yes No No
Tube type must be carefully matched with rotor type to prevent sample loss and/or tube failure
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)
30
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)
top related