AAC_Chapter 3 Centrifugation English_20100325

8/4/2019 AAC_Chapter 3 Centrifugation English_20100325

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Chapter 2 Centrifugation:

Separation of Organelles and Biomolecules

3.1 Introduction

.

3.3 Types, care and safety of centrifuges

.influenza virus and protein complex)

.

na y ca oc em s ry

3.4.3 Ultracentrifugation

1

, ,


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General Steps in Biochemical Separation

2


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Separation of Macromolecules (CAB)

roma ograp y, prec p a on Electrophoresis, ultracentrifugation

3


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Introduction (MBM 3.1)

A centrifuge is a device for separating particles from a

, , ,of the medium and rotor speed

In a solution, particles whose density is higher

particles that are lighter than it float to thetop. The greater the difference in density, the

.

density (isopyknic conditions), the particles

hover. To take advantage of even tinyerences n ens y o separa e var ous

particles in a solution, gravity can bereplaced with the much more powerful

4

centrifugal force provided by a centrifuge.


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Type 1: Analytical Centrifugation

supermolecular moleculesIn skeletal muscle, dystroglycan is acomponent of the dystrophin-

. . -Dystroglycan is an extracellular

peripheral membrane glycoprotein

1.

binding to a transmembraneglycoprotein, -dystroglycan. The -

- -

widely expressed in a broad array oftissues and is thought to stabilize thelasma membrane b actin as an

axis through which the extracellularmatrix is tightly linked to cytoskeleton.

This is because -dystroglycan stronglybinds to laminin in the extracellularmatrix, and the cytoplasmic domain of-dystroglycan interacts with dystrophin,

5http://www.cgmh.org.tw/chldhos/intr/c4a90/new_page_50.htm

which in turn binds to the actincytoskeleton2.


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Type 2: Preparative Centrifugation

, ,membrane vesicles

Lipid rafts ()

p ra s that float freely within the liquid-disordered bilayer of cellular

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Rafts are receiving increasing attention as devices that regulatemembrane function in eukaryotic cells.


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Simons, K. et al. J. Clin. Invest. 2002;110:597-603

Raft Proteins Are Targets fro Disease

Alzheimer diseaseParkinson diseaseHypertension

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3.2 Basic Principle of Sedimentation (AB 3.4.3)

Centrifugal force rMF 2=

M: mass of particle

r: radius of rotation (cm) (iestance o part c e rom ax s

of rotation)

-1

:Average angular velocity(radians/sec)

60 =

(r.p.m.)

8

revo ut on = ra ans

=360


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Centrifugal Field

G=r2 depends on the radical distance of the particle from the

( ) rminrev421-2

3600

9


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-1

60

= . . .

10


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Relative centrifugal force (different radius of rotor)

1-22

g

c grMg

rM

f

fRCF ===

/, "No. x g"

multi les of earth's ravitational force

1-

2

gr60

rmp2

RCF

=

().

RCF =1.12 x 10-5 x (rpm)2 x r

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Relative centrifugal force

RCF =

1.12 x 10-5 x (rpm)2 x r

rmin

rmax

12


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Interacting Forces in Centrifugation

Sedimentin force M2r is o osed b ...Fcentrifuge

1. Frictional Resistance against .= f.v

f = frictional coefficient of particle in the solvent

friction + buoyancy

=

2. Flotation Force rMsF2=

Ms = the mass of equal volume of solvent

2

BALANCE between the sedmenting force and counteracting force

M, f: relate to the mass

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-- spand shape of analyte


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Sedimentation Coefficient (s),

2

()p- s -

a e o e men a on

Svedberg unit

Theodor Svedberg(1884-1971)1926 Nobel prise

141908,


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rF 2=: massM

vrFcen

2= rotationofcenterformdistance:

oc yangu ar ve:r

fvFrict = articleofvolumes ecificarticle:densitysolvent:

v

FFF frictbuoycent +=tcoefficienfrictional:f

fvrMv /)1(2 =

rvS / 2= S:

fvMS b /)1( =

15


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M= particle massf= frictional coefficient of the particle in the solvent

= density of solution

v = particle velocity 3 S is increased for particle oflarger mass

2 ,

ecause se men ng orce a -vr

S is increased for particle oflarger density (equal volume)

equal particle mass (frictional coefficient is less)

S is increased with rotational speed

Mild, non-denaturing procedure is useful for protein

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pur ca on, an or n ac ce s an organe es


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M= article mass

f= frictional coefficient of the particle in the solvent

= density of solutionv = particle velocity2 particle specific volume (cm3/g, )

,2 ) ,

2

=1 (=),

17


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Separation by Sedimentation

100 kg 10 kg 110 kg30 kg 8Weight

Material Iron Stone Iron Stone Cotton Iron

8 ass

Densitytion

sity

30 kg10 kg Shape

enta

erde

100 kg

10 kg1

Sedi

High

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Densities and sedimentation coefficients for

biomolecules, cell organelles, and viruses.

density media

Hi h concentratedCsCl

19


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ion

menta o u eprotein

Sedi DNA

RNA

20


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21


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NOMOGRAMS

Conversion

between

relative

force

Equation used to calculate NOMOGRAMS (BMB Fig. 3.1)for quickly finding RCF at given speed and rotor type

.

22Radical distance(mm)

Relative centrifugalfield (xg)

Rotor speed (r.p.m)


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BMB 3.3.1

Maximum speed of sedimentationPresence /absence of vacuum

Volume of sample and capacity of

centrifugation tubes


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Microfuge

0.5-1.5 cm3, 10,000 g

Concentration of protein samples

Large-capacity preparative centrifuge

- cm , , - , g

24


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Hi h-s eed refri erated centrifu e

5-250 cm3, 100,000 g

eren a on separa on o nuc eus,

mitochondrial, protein precipitate, large

,

Ultracentrifugation5-250 cm3, 600,000

Microsomal vesicles, ribosome

Has to reduce excessive rotor temperature

generated by frictional resistance

25sealed chamber, evacuated, cooling


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Centrifuge Rotors (MBM3.3.2)

Fixed Angle Rotor Swinging Bucket Rotor

Lon er distance of travel ma allow

short distance to travel before

pelleting. Shorter run time.

better separation, such as in density

gradient centrifugation. Easier to

e mos w e y use ro or ype.disturbing pellet.

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Centrifuge Rotors (MBM3.3.2)

Vertical Tube Rotor

Swin inBucket Rotor

27


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. .On December 16, 1998, milk samples were

-

using a large aluminum rotor . The rotor failed

due to excessive mechanical stress

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Mechanical stress

Always ensure that loads are evenly balanced before a run.

Always observe the manufacturers maximum speed and sample

density ratings.

Always observe speed reductions when running high density solutions,p as c a ap ers, or s a n ess s ee u es.

Man rotors are made from either titanium or aluminum allo chosen

Corrosion

for their advantageous mechanical properties. While titanium alloys are

quite corrosion-resistant, aluminum alloys are not. When corrosion

,centrifugal force exerted during operation. The combination of stress and

corrosion causes the rotor to fail more quickly and at lower stress

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Differential Centrifugation BMB 3.4.1

Based on the differences in the

of different size, shape and density

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Moving Zone (differential) Centrifugation

-

Incomplete sedimentation (lower operation speed).

on ro me an so u on ens y

Mostly used for separation with similar density and

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different size/shape


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Moving Boundary (differential) Centrifugation

1) 3)

2)

1) The entire tube is filled with sample and centrifuged2) Through centrifugation, one obtains a separation of two

supernatant or in the pellet or it may be distributed in bothfractions, depending upon its size, shape, density, and

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con ons o cen r uga on

3) Repeat sedimentation at different speed


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Advantages: Large-scale preparation

sa van ages:

Poor resolution. Poor purity

33Difficult to separate analytes with similarsedimentation coefficient


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Density Gradient Centrifugation (BMB 3.4.2)

Important technique for purifying proteinsan par cu ar y nuc e c ac s.

Two different types of density gradient centrifugation, fortwo different ur oses are:

Zonal (or Rate Zonal) Centrifugation(Sucrose density gradient centrifugation)

-(Caesium chloride density gradient centrifugation)

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M i Z (diff ti l) C t if ti


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Moving Zone (differential) Centrifugation

1)

-

2)

1) Sample is applied in a thin zone at the top of the centrifuge

2).Under centrifugal force, the particles will begin sedimenting

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through the gradient in separate zones according to their size,

shape and density

Iso density (Isopycnic) Centrifugation


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Iso-density (Isopycnic) Centrifugation (AB3.4.3)Isopycnic = same density

= density equilibrium between analyte and solution

Both analytes and solution have different densities

1) Preparation of solution with different densities

E.g. sucrose:

oo water so u ty or ma ng g

concentration of solution

36 Cheap

Iso density (Isopyncic) Centrifugation


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Iso-density (Isopyncic) Centrifugation (AB3.4.3)

2). molecule floats or sinks to position where density equals density of

. .

and separation is on basis ofdifferent densities of the particles.

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Molecules separated on equilibrium position, NOT by

rates of sedimentation.

Comparison of Two Methods


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Comparison of Two Methods

IsopynciccentrifugationMoving ZoneCentrifugation

Centrifugation: Lower speed, notcomplete sedimented,

Completely sediment to wherethe density is equilibrated, high

,

Sedimentation Rate Sedimentation equilibrium

Similar MW,different density

Sample: Similar density,different MW/shape

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rote n s m ar ens ty,

but different in MW)

uc e c ac

cell organelle

Density Gradient Centrifugation


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Density Gradient Centrifugation

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The Influenza A Virus

Studded in the lipid bilayer are two integral

membrane proteins

some 500 molecules ofhemagglutinin ("H")an

some 100 molecules ofneuraminidase ("N")

Within the lipid bilayer are

some 3000 molecules ofmatrix protein

8 pieces of RNAEach of the 8 RNA molecules is associated

with

a globular particle (about 100 nm in diameter)

sheathed in a lipid bilayer (derived from the

many cop es o a nuc eopro e n

(2) several molecules of the three subunits

of its RNA polymerase

p asma mem rane o s os(3) some "non-structural" protein molecules

of uncertain function


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Four commonly used methods

Differential centrifugation and density

gra en cen r uga on

Precipitation of viruses

Denaturation of contaminants

Enz matic di estion of cell constituents

H t if i ?


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How to purify virus?

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Purification of influenza A virus (H1N1) by


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Purification of influenza A virus (H1N1) by

1. After 48 hours of incubation the allantoic fluidcontaining virus was purified by low speedcentrifugation (4000 rpm 40 min).

.18000rpm for 1.5 h in the SW27 rotor. The viruspellet was collected, diluted in STE buffer and

prepared in STE buffer.-----Isopyconic gradient centrifugation:():

. amp e recentr uge at rpm n t erotorfor 2.5 h at 4 C, then the virus band was

collected, diluted with STE buffer and pelleted bycentrifugation at 30000 rpm for 1.5 h at 4 C. Thepellet of

. .

BioMarket Ltd. www.biomarket.fi


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Sarcolemma ( :


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Sarcolemma ( :

45Skeletal Muscle


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How can we do separation?


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How can we do separation?

Step 1

Step 2

Step 3

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Step 1- Cell Homogenization (


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Step 1 Cell Homogenization ()

To obtain pure organelles, the cellsmust be ruptured, so that the cell

membrane is broken, but the

organelle to be studied is not. The

process o rup ur ng a ce s nownas homogenization of the cell and

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organelles is called fractionation.


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R t d ll d i


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Ruptured cells producing a

liquified cellularhomogenate

50


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Step 3- Density Gradient Centrifugation


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Sarcolemma

52


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ep - o ec on o rac ons

Manual collection by pipette

Automatic fraction collector for unstable

Freezing and slicing

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Electron

micro ra hs ofindividual myosinprotein molecules

Myosin is a major component of the contractileapparatus of muscle. As shown here, it is composed

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o wo g o u ar ea reg ons n e o a common

rodlike tail.

Hydrophobic and Low Abundant Membrane Proteome


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Hydrophobic and Low Abundant Membrane Proteome

Immuno-modulation, Molecular recognition , Cell surface adhesion

Heterogeneous

P

PP

P P

P

P

P

PP

Cell

CycleP P

PPDynamic,

Spacious

PhosphorylationSignal transduction Differentiation,

proliferation

Protein degradation

Apoptosis

55

Affinit Purification of Membrane


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Affinit Purification of Membrane

Vesicles (BMB 3.4.5)

Cross-contamination of

vesicular membrane

Right-side-outIn-side-out

protein

Inside-out vesicles,

right-side-out vesicle,

membrane sheet, leaky

Smaller vesicles are

vesicles

In-side-out c to lasmic side out

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Right-side-out (apoplastic side out)

vesicles

Lectin Agglutination Method


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-

Lectin: protein that interact with carbohydrate

57

There are many carbohydrates on the surface of

cell


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Lectin Agglutination Method


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WGA: Wheat germ agglutinin

SL: Sarcolemma

No carbohydrate

59


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Analytical Ultracentrifugation (AUC)


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. .

An analytical ultracentrifuge spins a rotor at an accuratelycontrolled s eed and tem erature. The concentration

distribution of the sample is determined at known times

using absorbance measurements. It can determine:

Purity of macromoleculeChan e in relative molecular mass of su ermolecular

complexes

Shape, Conformational change of protein structure

Ligand-binding study

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(Beckman Optima XL-A):Optical System of an Analytical


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This figure displays a schematic

Optima XL-A absorbancesystem. A high intensity xenon

as amp a ows e use o

wavelengths between 190 and

800nm. The lamp is fired briefly

as a selected sector passes the

detector.back to top

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Sedimentation Velocity Method


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Mass, Shape and Conformation; Higher velocity

are performed at high speed to

overcome the effect of diffusion. For a

Sedimentationsedimentation velocity experiment, an

initially uniform solution is placed in a

cell and a sufficiently high angular

velocity is applied to cause rapid

sedimentation of solute towards thecell bottom. As a result there is a

depletion of solute near the meniscus,

causing a characteristic spectrum asDiffusion .

boundary occurs between the depleted

region and the sedimenting solute (the

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Determination of Sedimentation Coefficient (s)


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4000 s

10000 s

8000 s

The velocity of the individual particles in SV experiments cannot be

resolved, but the rate of movement of the boundary region can be

. ,determined. Remember, sdepends directly on the mass of the soluteparticles and inversely on the frictional coefficient, which is a

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measure o s ze o e so u e par c es.

http://www-bioc.rice.edu/bios576/AU/AU_Page.html#au

Sedimentation Equilibrium Methods


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Mass, Complex formation; Lower velocity e men a on equ r um

experiments have a lower rotorspeed than sedimentation

velocit ex eriments. Soluteparticles do not pellet at thebottom of the cell, but instead theprocess ofdiffusion opposes theprocess o se men a on un

after a period of time, the twoopposing forces reache uilibrium and the a arentconcentration profile does notchange. At equilibrium, theconcentration of the solute

the cell bottom. Each columndisplays a different absorbancerofile because the

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concentrations of sample are

varied in each.

Sedimentation Analysis of


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The binding of ligands may induce

con orma ona c anges n su un s o

biomolecules, which changes the

supramolecular structure of complex.

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AAC_Chapter 3 Centrifugation English_20100325