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)