electrophoresis editted

8/13/2019 Electrophoresis Editted

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ELECTROPHORESISDone By: Samyah Alanazi


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lecture Outline

1- Introduction.

2- Principle.

3- Factors affecting the rate of migration .

4- solutions. 5- Principal of electro-endosmosis.

6- Factors affecting electrophoresis mobility.

7- Instrumentation.

8-Types of electrophoresis.


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Introduction

The term electrophoresis describes the migration of a

charged particle under the influence of an electric field.

Many important biological molecules, such as amino acids,peptides ,proteins ,nucleotides and nucleic acids,

possess ionisable groups and, therefore, at any given

pH, exist in solution as electrically charged species either

as cations (+) or anoins (-).

Under the influence of an electric field these charged

particles will migrate either to the cathode or to the anode,

depending on the nature of their net charge.


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Principle

Any charged ion or molecule migrateswhen placedin an electric field.

Therate of migration depend upon its net charge,

size, shape , pH , properties of the supportmedium, time frame of the procedure, temp. of theoperating system and the applied electric current.

This can be representedby following eq.:

E * q

V = ---------------

f


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v = velocity of migration of the molecule. E = electric field in volts per cm q = net electric charge on the molecule f = frictional coefficient

The movement of charged particle in an electric field isexpressed in terms of electrophoretic mobility ,denotedby .where,

= q/fFor molecules with similar shapes, f doesnt varyhowever, it varies with size. Thus electrophoretic mobility() of a molecule is directly proportional to chargedensity(charge\mass ratio).


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The electric field is removed before the molecules in thesample reach the electrodes, the components will havebeen separated according to their electrophoretic mobility.Thus, electrophoresis is an incomplete form of electrolysis

. The sample then are then located by staining with anappropriate dye.

The current in the solution between the electrodes isconducted mainly by the buffer ions, a small proportion

are being conducted by the sample ions. Ohms lawexpress the relationship between current (I),voltage (V)and resistance ( R ):

R = V/I.


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It is therefore appears that it is possible to accelerate an

electrophoretic separation by increasing the applied

voltage, which would result in a corresponding increase

the electric flowing . The distance migrated by the ions will

be proportional to both current and time.


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FACTORS AFFECTING MIGRATION

RATE1- Net Charge higher the charge greater theelectrophoretic mobility. It depends on buffer pH andPI.

2- Size bigger the molecule greater are thefrictional and electrostatic forces exerted on it by themedium. Consequently, larger particles have smallerelectrophoretic mobility compared to smallerparticles.

3- Shape rounded contours elicit lesser frictionaland electrostatic retardation compared to sharpcontours. Therefore globular protein move fasterthan fibrous protein.


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4- Buffer pH: it has been chosen in order to maintain netcharge of a molecule/atom.

5- prosperities of support medium:

A higher pore size will increase mobility while, increaseviscosity will slower the movement.

6- time- frame of the procedure:

longer time: Sample would've ran off the gel.

shorter time: not far enough and would have caused thebands to not separate enough, being merged and unable to beread

7- Temp. of the system:

Increase heat lead to the followings:


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Heat effects on the particle mobility

A- An increased rate of diffusion of sample and bufferions leading to broadening of separated sample.

B- the formation of convection currents, which leads to

mixing of separated samples.

C-Thermal instability of samples that are rather sensitiveto heat. This may include denaturation of proteins (and

thus the lose of enzyme activity).

D- A decrease of buffer viscosity, and hence a reductionin the resistance of the medium.


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solutions

1- Using a stabilised power supply which provides

constant power and thus eliminates fluctuations in heating

.

8- final factor affecting separation is called

electroendosmosiswhich is due to presence of charged

groups on the surface of support medium .


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Principal of electro-endosmosis

The static support, the stabilizing medium (e.g. the gel)

and/or the surface of the separation equipment such as

glass plates, tubes or capillaries can carry charged

groups: e.g. carboxylic groups in starch and agarose,

sulfonic groups in agarose, silicium oxide on glass

surfaces. These groups become ionized in basic and

neutral buffers: in the electric field they will be attracted by

the anode. As they are fixed in the matrix, they can not

migrate. This results in a compensation by the counterflow of H3O

+ ions towards the cathode:

electroendosmosis.


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In gels, this effect is observed as a water flow towards the

cathode, which carries the solubilized substances along.

The electrophoretic and electroosmotic migrations are

then additive. The results are: blurred zones, and drying

of the gel in the anodal area of flatbed gels.

When fixed groups are positively charged, the

electroosmotic flow is directed towards the anode.


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Instrumentation

1- Power back which supply DC.

2- Electrophoresis unit (for running either horizontal or

vertical ) : two glass plates (different pore sizes), plasticspacers, buffer and comb.


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TYPES OF ELECTROPHORESIS

ACCORDING TO THE TYPES OF

SUPPORT MEDIUMELECTROPHORESIS

FREE

ELECTROPHORESIS

ZONE

ELECTROPHORESIS

MICRO

ELECTROPHORESIS

MOVING

BOUNDARY

PAPER

ELECTROPHORESIS

CELLULOSE ACTTATE

ELECTROPHORESIS

GEL

ELECTROPHORESIS


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PAPER ELECTROPHORESIS

It is the form of electrophoresis that is carried out on filterpaper. This technique is useful for separation of smallcharged molecules such as amino acids and smallproteins.

FILTER PAPER : It is the stabilizing medium. We can useWhatman filter paper, cellulose acetate filter paper orchromatography paper.


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Cellulose acetate electrophoresis

One of the older methods but it still has a number of

applications.

It has an advantage over paper in that it is a much more

homogeneous medium, with uniform pore size, and does

not absorb proteins in the way that paper does.

It gives better resolution than paper electrophoresis

,however nothing as good as that achieved with

polyacrylamide gels.

It is especially useful for separating alpha immunoglobulins

from albumin.


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GEL ELECTROPHORESIS

It is a technique used for the separation of Deoxyribonucleicacid, Ribonucleic acid or protein molecules according to theirsize and electrical charge using an electric current applied to agel matrix.

What is a gel?Gel is a cross linked polymer whose composition and porosity ischosen based on the specific weight and porosity of the targetmolecules.

Types of Gel:1- Starch gel

2- Agarose gel.

3- Polyacrylamide gel.


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Starch gel :Anearly form of gel .

Agarose gel :A highly purified uncharged polysaccharide

derived from agar.

Used to separate macromolecules such as nucleic acids,

large proteins and protein complexes. Percentage used

(1-3%).

Polyacrylamide gel (PAGE) : Used to separate most

proteins and small oligonucleotides because of the

presence of small pores. Percentage used (3-30%).


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Example:

PCR products of the C-terminus of killifish CFTR gene before

and after PCR purification. Both products were DNAelectrophoresed in 3% Agarose gel. Bands in the first lane and third

lane are referring to PCR product before and after PCR purification

respectively. An estimation of PCR products sizes was determined by

using DNA 100 bp marker in the second lane.


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Sodium dodecyle sulphate (SDS) polyacrylamide gel

electrophoresis.

The most commonly used technique for the separation of proteins isSodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS PAGE). Other used gels are: native gel, gradient gel and IFEgel, cellulose acetate elect. and 2D-polyacrylamide gelelectrophoresis.

PROCEDURE

Protein sample is first boiled for 5 mins in a buffer solutioncontaining SDS and -mercaptoethanol.

Protein gets denatured and opens up into rod-shaped structure.

Sample buffer contains bromophenol blue which is used as atracking dye, and sucrose or glycerol.

Before the sample is loaded into the main separating gel astacking gel is poured on top of the separating gel.


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Cont.

Current is switched on.

The negatively charged protein-SDS complexes now

continue to move towards the anode.

As they pass through the separating gel, the proteins

separate, owing to the molecular sieving properties of the

gel.

When the dye reaches the bottom of the gel, the current is

turned off.

Gel is removed from between the glass plates and shaken

in an appropriate stain solution.

Blue colored bands are observed under UV rays.


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Example

The Purification check of the C-terminus of Killifish CFTR.

Purification was checked using 15% SDS-PAGE gel. The

Prestained Protein Marker in the first lane was used for molecular

weight determinations. The lanes from (2-5) refer to IMAC unbound

materials; washes and elutions before thrombin cleavage while

lanes from (6-9) refer to IMAC unbound materials; washes and

elutions after thrombin cleavage. The arrow in lane 6 refers to the

purified c-terminus peptide.


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TWO-DIMENSIONAL

ELECTROPHORESIS This technique combines the technique IEF (first

dimension), which separates proteins in a mixtureaccording to charge (PI), with the size separationtechnique of SDS-PAGE second dimension).

The combination of these two technique to give two-dimension(2-D)PAGE provides a highly sophisticatedanalytical method for analysing protein mixtures.


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Detection of components after

electrophoretic separation 1- Proteins:

A- Comassie Brilliant blue.

B- Silver protein.

C- Instant blue.

2- protein (western blotting):

It involves transferring of proteins from gel to nitrocellulosepaper to produce a sandwich of gel and nitrocellulose thatcompressed in a cassette and immersed in a buffer between

parallel electrodes. when the current is passed it causeseparating of protein from gel to nitrocellulose sheet which willexamine further by using antibodies (specific to tested protein) ,incubation ,second antibodies with marker and then exposeblot to UV light.


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Capillary electrophoresis

Used to separate wide range of biological moleculed

including : A.A , peptides, proteins, DNA fragments , metal

ions and drugs.

It involves electrophoresis of sample in a very narrow-

bore tubes.

It is a highly speed analysis (20 mins ).

It is a type of zone electrophoresis.


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Microchip electrophoresis Very high speed analysis (tens of seconds).

Use laser induced fluorescence.

It required very low volts.


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Clinical applications

1- Specific protein electrophoresis.

2- Identification and quantification specific serum protein

classes.

3- Separation and quantification of lipoprotein and lipid

classes.

4- Separation and quantification of enzymes to its

subtypes.

5- Separation and quantification of immunoglobulins.

6- Use of western blot tech./ southern blot tech.

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