Electrophoresis. .pptx

ELECTROPHORESIS
BY :- DHRUMI NAIK

 Electrophoresis is a physical method of analysis which involves separation of the compounds
that are capable for obtaining electric charge in conducting electrodes.
 Electrophoresis is the movement of charged particles through an electrode when subjected to
an electric field.
 By this technique solutes are separated by their different rates of travel through an electric
field.
 Commonly used in biological analysis, particularly in the separations of proteins, peptides and
nucleic acids.
INTRODUCTION

 Electrophoresis may be defined as migration of charged particles through solution under
influence of external electrical field.
 Ions that are suspended between two electrodes tends to travel towards that electrodes that
bears opposite chargers.
 Cations move towards cathode
 Anions move towards anode
DEFINITION

PRINCIPLE
 The migration velocity “V” of ions in electric field is equal to product of field strength “E”’
Electrophoretic mobility µ.
V =E * µ
 Electrophoretic Mobility = Ionic Strength
Frictional Retarding Factor
 If two species differ in charge or either frictional force they get easily separated from each
other but neutral species are not separated.

 Electrophoretic Mobility
Electrophoretic mobility is defined as the rate of migration (cm/sec) per unit field strength
(volts/cm).
µ = Q / 6πrη
Where µ = Electrophoretic Mobility
Q= Net charge of the ion
r = Ionic radius of solute
η = Viscosity of medium

 The electrophoretic mobility is directly proportional to net charge and inversely proportional
to molecular size and viscosity of the electrophoresis medium.
 The pH of solution affects the mobility of the ion by determining the amount and nature of
charge.
 Proteins, nucleic acids, nucleosides and amino acids bear charged polar groups making them
suitable group for electrophoresis.
 Carbohydrate carrying no charges groups are first bound to charged groups like Borate or
Sulfite ions and then electrophoresis is carried out.
 Lipids are not electrophoresed because electrophoretic current requires polar solvents in
which most lipids are insoluble.

TYPES OF ELECTROPHORESIS
1. ZONE ELECTROPHORESIS
 Paper Electrophoresis
 Gel Electrophoresis
 Thin Layer Electrophoresis
 Cellulose Acetate Electrophoresis
2. MOVING BOUNDARY ELECTROPHORESIS
 Capillary Electrophoresis
 Iso - Electrophoresis

ZONE ELECTROPHORESIS
 It involves the migration of the charged particles on the supporting media.
 Paper, Cellulose acetate membrane, Starch Gel, Poly acrylamide.
 Components separated are distributed into discrete zone on the support media.
 Supporting media is saturated with buffer solution, Small volume of the sample is applied as
narrow band.
 Zone electrophoresis is an electrophoretic separation technique used for analyzing proteins,
nucleic acids, biopolymers.

 Advantages :-
 Useful in biochemical investigation
 Small quantity of sample can be analyzed
 Low cost and easy maintenance
 Disadvantage :-
 Unsuitable for accurate mobility and isoelectric point determination.

1. PAPER ELECTROPHORESIS
 Paper electrophoresis comes under zone electrophoresis.
Principle :-
 When charged molecules are placed in an electric field they migrate towards either
positive or negative pole electrode according to their charge.
 In contrast to protein which can have either a net positive or net negative charge.
 Nucleic acids have a consistent negative charge imparted by their phosphate backbone,
and migrate towards the anode.

Equipment :-
 The equipment required for electrophoresis consist a basically of two items, a POWER PACK
and ELECTROPHORETIC CELL.
 POWER PACK :-
 Power pack provides a stabilized direct current and has controls for both voltage & current
out put, which have an out put of 0 to 500V and 150 mA are available.
 THE ELECTROPHORETIC CELL :-
 It contains : The electrodes, buffer reservoir, a support for paper and a transparent
insulating cover.
 The electrodes are usually made of platinum.

Working :-
 A long strip of filter paper is moistened with a suitable buffer solution of desired pH and the
sample is applied transversely across the central part of the strip.
 Ends are fixed to dip in buffer solutions in two troughs fitted with electrodes.
 Electric field of about 20 volts/cm is established.
 The charged particles of sample migrate along the strip towards respective electrodes of
opposite polarity, according to net charges, sizes and interactions with the solid matrix.
 Homogeneous groups of particles migrate as a separate band.
 The electrophoresis is carried out for 16-18 hours.
 Proteins are stained (bromophenol blue) to make them visible.
 The separated proteins appear as distinct bands.

Types of paper used :-
 Whatmann paper no. 1,2,3 etc.,
 Eaton – Dikeman 301-85, 320 and 352.
 Munktells 20/50
 Large sheet of whatmann thin paper 1 for large volume sample
Types of paper electrophoresis
 Horizontal paper electrophoresis
 Vertical paper electrophoresis
 Continuous paper electrophoresis

 In vertical mode the separation and migration of ions is assisted by gravity in addition to
electrophoretic mobility.

 In this method the thin sheet of filter paper is usually used as supporting medium.
 It is used for preparative sample.

 Horizontal paper electrophoresis in which strip is positioned in apparatus without any
distortion and lower part is immersed solution.
 separation is achieved in 12-14 hours.

 APPLICATION
 Paper electrophoresis has emerged as a simple, inexpensive, and accurate lab procedure for
various research and clinical studies.
 Clinical Application of paper electrophoresis include study of sickle cell disease, hemoglobin
abnormalities, separation of blood clotting factor and serum plasma protein from blood
sample.

 It has also been used in separation and identification of alkaloids.
 Paper electrophoresis also be used for testing water sample, toxicity of water and other
environmental components.
 Drug testing industries used PE to determine presence of illegal drugs crime suspects.

2. GEL ELECTROPHORESIS
 Gel electrophoresis is a method for separation and analysis of macromolecules like DNA, RNA
and proteins of their fragments, based on their size and charge.
 Gel electrophoresis uses gel as an anti-convective medium or sieving medium during
electrophoresis.
 By placing the substance to be separated in wells of the gel and applying an electric current,
allows the molecule to move through the matrix at different rates towards the anode if
negatively charged or toward the cathode if positively charged.

 As they move through the gel, the larger molecule will be held up as they try to pass
through the pores of the gel, while the smaller molecules will be impeded less and move
faster.
 This results in a separation by size, with the larger molecules nearer the well and the
smaller molecules father away.

Principle of Separation
 According to charge :-
When charged molecules are placed in an electric field, they migrate toward either the positive
(anode) or negative (cathode) pole according to their charge.
 According to size :-
The smaller molecules move more swiftly than the larder sized ones, as they can travel through
pores more easily .

Instrumentation
 Electrophoresis apparatus
 Buffer
 Power supply
 Supporting media
 Detection and Quantification

 Electrophoresis Apparatus
 The casting tray is made up pf plastic or glass.
 The comb contain varying number of teeth which is used for formation of well in proper manner
desire for separation.

Electrophoresis Apparatus Set up

 Buffers
 Buffers in gel electrophoresis are used to provide ions that carry current and maintain pH
at relative constant value.
 Buffers usually used are EDTA / TAE

 Power supply
 Electrodes are connected to their respective terminal of electrophoresis chamber and to
power supply with control rate of current flow.
 The best resolution of fragment larger than about 2 kb is attained by applying nor more than
5 V/CM to the gel.

 Supporting Media (GEL)
 Starch
 Agarose
 Cellulose acetate
 Polyacrylamide Gel
 The kind of separating matrix depends on the type of molecule to be separated.
 Agarose and polyacrylamide gels are crossed linked and forms sponge like structure.
 It is important that supporting media should be electrically neutral as presence of charge may
cause molecule migration retardation.
 Agarose gel have large pore size and used for separating larger DNA molecule and
polyacrylamide gel is used to obtain high resolution separation for smaller DNA molecule.

 Detection and Quantification
1. Stains
 Protein staining
 Ethidium bromide staining
2. Blotting
 Southern blotting (for DNA)
 Northern blotting (for RNA)
 Western blotting (For protein)

Process of Gel Electrophoresis

 Visualization
 The molecules in the gel are stained to make them visible.
 DNA may be visualized using ethidium bromide which, when intercalated into DNA,
fluoresce under UV light, while protein may be visualized under silver stain.
 SYBR Green I is more expensive, but 25 times more sensitive and possible safer than
ethidium bromide.
 SYBR Safe is a variant of SYBR Green, and show low level of mutagenicity and toxicity.
 Other less frequently used markers are cresol red and orange G.

APPLICATION :-
 Application of gel electrophoresis in estimation of size of DNA molecules and investigation of DNA
cleavage efficiency of small molecules, for example are extensively used in molecular biology
 Gel electrophoresis is also commonly used in plant breeding and genomics.
 Separation of ribonucleic acid
 Separation of amino acid
 Separation of antibiotic drug
 Separation of lipoproteins
 Separation of enzyme in blood
 Separation of RNA and DNA
 Separation of protein molecules

FACTORS AFFECTING SEPARATION
1. The Sample
 Charge :- Higher the charge greater the mobility
 Size :- Bigger the molecule greater the frictional and electrostatic force exerted on it by the
medium i.e. Larger particles have smaller electrophoretic mobility compared to smaller particles.
 Shape :- The globular proteins will migrate faster thafttn the fibrous protein.
MORE MOBILITY

2. Electric field
 Increase of migration with the increase of voltage gradient.
3. Buffer
Migrate of charge particles depend on the buffer.
a) Composition
Commonly used buffers are “Formate”, ‘’Acetate’’, “Citrate”, “Phosphate”’, ‘’EDTA”.
The choice of buffer depends upon the type of sample being electrophoresed.
b) pH
The extent of ionization depends upon pH, especially in organic compounds.
The ionization increases in pH of organic acids and its just reverse for the organic bases therefore
affecting its rate of migration.

3. The Medium
 The inert medium can exert adsorption, molecular sieving effects & electro – osmosis
– processes that affect the electrophoresis rate.
Adsorption :-
 It means retention of a component on the surface of supporting medium.
 The rate and resolution of the electrophoretic separation can be efficiently reduced by
adsorption.
 Molecular Sieving :-
 Media such as “Polyacrylamide”, “Agar’’, “Starch”’, & “Sephadex” have cross – linked
structures giving rise to pores with in gel beads.

4. Heat Generation in electric fields
 One of the practical problems encountered in electrophoresis is generation of heat
from resistance in the electrophoretic medium.
 Heating not only changes viscosity and density of the electrophoretic media, It also
damage equipment.

Electrophoresis. .pptx

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