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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.