Sedimentation andSedimentation andSedimentation and

ElectrophoresisElectrophoresis

Biophysics seminarsBiophysics seminars

Analysis and separation of macromoleculesmacromolecules

Physical parameters Methods

1. Molecular mass Sedimentation velocity method

2. Size Gel electrophoresis2. Size Gel electrophoresis

3. Shape Sedimentation velocity method

4. Density Density gradient centrifugation4. Density Density gradient centrifugation

5.Electrical charge Isoelectric focusing

6. Nucleotide sequence Southern blotting6. Nucleotide sequence Southern blotting

7. Presence of specific binding site Western blotting

SedimentationSedimentation

SedimentationSedimentation

Why is the centrifugation important?

In general, the aim is to describe the size or the mass of the particles.The methods based on gravitation are effective only in the range of 2-50 µm particle radius.The methods based on gravitation are effective only in the range of 2-50 µm particle radius.In the case of smaller molecules the sedimentation is based on the method of centrifugation.

Theodor Svedberg (Swedish chemist, Nobel-laureate in 1926):Theodor Svedberg (Swedish chemist, Nobel-laureate in 1926):

the first ultracentrifuge (N>10000 min-1)

Ultracentrifuge

Preparative Analytical

Separation of different size and

different mass of components

Size or molecular mass

determination

What do the forces affect toWhat do the forces affect to

the particles?

How to calculate the forces?

m, ρ0

Centrifugal force: f vFrictional force:

How to calculate the forces?

m, ρ0

2ma mr= ω Buoyant force:2

0 0

mVg rρ = ρ ω

ρ

How long is the particles accelerating?

F =F -F

It can accelerate while:

Ffrictional=Fcentrifugal-Fbuoyant

Sedimentation velocity method

bcf FFF −=0=++ buoyantfrictionallcentrifuga FFF

Sedimentation velocity method

In equilibrium:

−=−=ρρωωρ

ρω 022

02 1mrr

mmrfv

Is then the velocity =constant?

The value of the centrifugal field depends on the radius, it increases further from the axis.

NO!

In the case of sedimentation the velocity of the particle increasesalso further from the axis. (velocity is place-dependant)

mv

−ρρ01

fr

vS

==

ρω 2

Sedimentation constant: S 1 Svedberg=10-13 s Theodore Svedberg (1884-1971)

Swedish chemist

Nobel-laureate in 1926

Why is it important or useful to us?Why is it important or useful to us?

We can calculate the mass of the molecule.We can determine the velocity and the centrifugal acceleration.We can determine the velocity and the centrifugal acceleration.

mv

S

−==

ρρ 01

Can be calculated.

D – diffusion constant

fr

vS ==

ρω 2

Can be measured. D – diffusion constant

k - Boltzmann constant

R – universal gas constant

N – Avogadro number 6*1023

Can be measured.

To determine the mass one needs to combine theTo determine the mass one needs to combine thesedimentation method with diffusion measurements.

Sedimentation equilibrium methodSedimentation equilibrium method

Particles are awaited to reach the bottom of thetube: their average sedimentation speed is 0. r1tube: their average sedimentation speed is 0.

A balance is set between the sedimentationand the thermal diffusion.

r1

r2and the thermal diffusion.

The energy of the thermal movement brings aportion of the particles into higher energy state.

Near the bottom of the tube

a given distribution of the

particles is present.portion of the particles into higher energy state.

particles is present.

At r1 and r2 distances from the axis the ratio of n1 and n2 concentrationsAt r1 and r2 distances from the axis the ratio of n1 and n2 concentrationscan be calculated from the Boltzmann-distribution:

E-potential energy of themolecules at the given r1 or r2point.

The difference of E1 and E2 potential energy:The difference of E1 and E2 potential energy:

The higher angular velocity – narrower the r interval in which themolecules are locatedmolecules are located

In equilibrium:

Can be measuredCan be calculated

The advantages of thismethodmethod

The mass can be calculated.The mass can be calculated.

Not need diffusion measurements.

The results are not depend on the shape constant.The results are not depend on the shape constant.

There is one problem:

Density determination

Density can be determined byDensity can be determined bydensity gradient centrifugation.

Density gradient centrifugationDensity gradient centrifugation

Centrifuging low molecular weight molecules with high density (e.g.Centrifuging low molecular weight molecules with high density (e.g.CsCl, CsBr) results in a density gradient upon reaching equilibrium.When macromolecules are centrifuged in such a medium they stopwhen reach same density medium.when reach same density medium.

Meselson-Stahl experiment

Differencial centrifugationDifferencial centrifugation

Differencial centrifugationDifferencial centrifugation

ExampleExample

You would like to separate one protein from the cell debris, thereforeYou would like to separate one protein from the cell debris, thereforeyou need to apply a centrifugal force of 15000 g. The radius of therotor is 10 cm. Which RPM would you use for UC to achieve theseparation?

RCF= 15000g a= 15000x10=150000g

aRCF cp=

separation?

rnra cp222 4πω ==

rNrN

acp2

22 011,0

604 =

= πn- revolutions per second

cp 60

n- revolutions per second

N-RPM

r-radius (m)a

r

aN cp

011,0=

ElectrophoresisElectrophoresis

Movement of charged molecule in

electrostatic field.electrostatic field.

ElectrophoresisWhat are the forces?

Electrophoresis

ZeE

v

+ -+Ze

ZeEfv

E

1. Coulomb-force 2. Frictional force

E

FC=ZeE

E=electrostatic field

Ffrictional = f v

E=electrostatic field

e=elementary charge

Z: the number of charges

v: velocity

f: shape factor

How long is the particle accelerating?

ZeE = fv

How long is the particle accelerating?

In equilibrium:

ZeE = fv

The electrophoretic mobility

lv = V

E =t

lv =

ld

d

VE =

el

ldu

Vt= l – the distance covered by a

particle (l) within a certain time

t – time (s)t – time (s)

V – voltage (V)

d – electrode distance (m)

Types of electrophoresisTypes of electrophoresis

I. Free electrophoresis

II. Capillary electrophoresis

III. Gel electrophoresis1. Agarose gel electrophoresis

2. Poliakrilamide gel electrophoresis2. Poliakrilamide gel electrophoresis

3. Gradient gel electrophoresis

4. Izoelectric focusing4. Izoelectric focusing

5. Two dimensional electrophoresis

IV. Blotting techniquesIV. Blotting techniques1. Southern Blot

2. NorthernBlot2. NorthernBlot

3. Western Blot

II. Capillary electrophoresisII. Capillary electrophoresis

The differential movement for migration of ions by attraction or repulsion in an electric field.attraction or repulsion in an electric field.

Depending on the charge, the molecules move through at different speeds.

capillarydetector

at different speeds.

• small size, big charge –bigcapillary

25-75 µm

inner diameter

• small size, big charge –bigmobility/fast• big size, small charge –smallmobility/slow

buffer

buffer

sample

mobility/slow

buffer

electrode

sample

electrodeelectrode

High voltage Detection: OD, λ = 200 nm

The basic of the method:

The elecroosmotic flow (EOF)

The elecroosmotic flow formation:inner surface of negative charge quartz capillary (Si-O-)

hydrated cations accumulate near by the surface

Double layer in the wall of

capillary hydrated cations accumulate near by the surfaceThe electric field causes mass flowing

capillary

Migration:

1. Cations

2. Neutral

molecules

3. Anions3. Anions

The advantages of this technique:

Law heat development

Short time, fast

Relatively inexpensive

Small sample (1-10 nl)

AutomatedAutomated

Examples

albumin

Normal levels of serum components

bétaα-1 α-2 gamma

Albumine consists of two fractions - rare bisalbuminaemia Higher α-1 and α-2 fraction – inflamed processAlbumine consists of two fractions - rare bisalbuminaemia

(metabolism disorder)

Higher α-1 and α-2 fraction – inflamed process

III./1. Agarose gelelectrophoresisIII./1. Agarose gelelectrophoresis

Nucleic acid molecules are separated by applying an electric field tomove the negatively charged molecules through an agarose matrix.move the negatively charged molecules through an agarose matrix.Shorter molecules move faster and migrate farther than longer onesbecause shorter molecules migrate more easily through the pores ofthe gel.

Agarose:

the gel.

Agarose:

Sample dyeing:

It consists of polysaccharide chains.

Sample dyeing:It can help the visibility of thesample until the running.

Etidium-bromide:

It fluoresces under UV light when intercalated into DNA (or RNA).intercalated into DNA (or RNA).

III/2. SDS- Poliakrilamide gelelectrophoresisIII/2. SDS- Poliakrilamide gelelectrophoresis

Acrylamide + bisacrylamide + persulfate → poliacrylamideAcrylamide + bisacrylamide + persulfate → poliacrylamide

The pore size of gel depends onthe concentration of monomers.

Components:

the concentration of monomers.Protein sepration method.

Components:

APS- initiates the polymerizationTemed- free radicals, which catalysesTemed- free radicals, which catalysesthe polymerisationBisz-akrilamid- crosslinkerSDS - anionic detergent: anionic polar end and a long apolar tail, denaturizing agentand a long apolar tail, denaturizing agentIt can bind the apolar part of the protein, makean electric interaction with the positive regions, the negative regions stay freeAll the proteins become negative separation of the molecules according to their All the proteins become negative separation of the molecules according to their size at a given pH (the small one is quicker, the big one is slower!)

III/4. Isoelectric focusingIII/4. Isoelectric focusing

Separate the proteins based on their charges, or Separate the proteins based on their charges, or more precisely based on their pI points.

Proteins contain both positive and negative charge Proteins contain both positive and negative charge groups.If the pH changes, their net charge is changing as well;well;at high pH-n: negative;at low pH: positive.

Isoelectric point: the pH value where the protein has zero net charge.

The electrophoresis is done in a pH gradient: the macromolecules move to their pI point and stop there loosing their net charge.there loosing their net charge.

III./5. Two dimensional electrophoresisIII./5. Two dimensional electrophoresis

The principal: two separation methods are applied and theyseparate the sample proteins by two different sets of properties.

Perpendicular running of the methods:Protein mixture

separate the sample proteins by two different sets of properties.

1. Separation by isoelectric focusing (based on pI points);

Isoelectricfocusing

mixture

Separation bycharge

2. SDS-PAGE gel electrophoresis(based on molecular weights).

Separation bysize

SDS electrophoresis

IV. A Southern, Northern and Western IV. A Southern, Northern and Western techniques

DNA, RNA and proteins can separated byDNA, RNA and proteins can separated by

electrophoresis.

The separated molecules can blotted to a The separated molecules can blotted to a

filterpaper.

HybridizationHybridization

Antibody staining – protein identification.

Southern- DNASouthern- DNA

Northern –RNA identification

Western – Protein

IV./1. Southern BlotIV./1. Southern Blot

1. DNA is digested with restriction

enzyme

Staining gel autoradiogram

enzyme

2. Agarose gel electrophoresis- we

get a lot of same size fragments.

3. Denaturation of DNA fragments

4. Blotting to a nitrocellulose4. Blotting to a nitrocellulose

membrane

5. Hybridization ( DNA is labelled

with specific DNA probe)with specific DNA probe)

6. The same length, but different structure fragments are visualised6. The same length, but different structure fragments are visualised

with autoradiography after the labelling.

IV./3. Western BlotIV./3. Western Blot

1. SDS-gel

2. Blotting to the nitrocellulose2. Blotting to the nitrocellulose

membrane

3. Making with primary

monoclonal antibody– specificmonoclonal antibody– specific

binding

4. Secondary antibody and it can

bind a catalytic enzyme →colourbind a catalytic enzyme →colour

changing

5. Enzyme – substrate adding

6. Sign detection6. Sign detection