Introduction to Electrophoresis

1. ELECTROPHORESIS
Dr. M. Sonia Angeline
Assistant Professor
Department of Life Sciences
Kristu Jayanti College

2. • This electro-kinetic phenomenon was observed for the first time in 1807 by Ferdinand
Frederic Reuss (Moscow State University), who noticed that the application of a
constant electric field caused clay particles dispersed in water to migrate.
• It is ultimately caused by the presence of a charged interface between the particle
surface and the surrounding fluid.
• It is the basis for a number of analytical techniques used in biochemistry for
separating molecules by size, charge, or binding affinity.
• Electrophoresis is a laboratory technique used to separate DNA, RNA, or protein
molecules based on their size and electrical charge.
• An electric current is used to move molecules to be separated through a gel.
INTRODUCTION

3.  Pores in the gel work like a sieve, allowing smaller molecules to move faster than
larger molecules.
 The conditions used during electrophoresis can be adjusted to separate molecules in
a desired size range.
 Electrophoresis is defined as the migration of charged ions in an electric field.
 In metal conductors, electric current is carried by the movement of electrons,
largely along the surface of the metal.
 In solutions, the electric current flows between electrodes and is carried by ions.
 The ions that migrate towards the anode, because of their anionic migration, are
called “anions”.
 The ions which will migrate to the cathode are called “cations”.
INTRODUCTION

4. PRINCIPLE
 When charged molecules are placed in an electric field, they migrate toward either the
positive or negative pole according to their charge.
 In contrast to proteins, 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 toward the anode.
 This force will cause the protein to accelerate towards either the cathode or the anode,
depending on the sign of its charge.
 Of course there are other forces such as frictional force when ions move in the electric
field.
 The influence of them cannot be understood easily.
 Electrophoresis exploits the fact that different ions have different mobility in an electric
field and so can be separated by this way.

5. • Proteins and nucleic acids are electrophoresed within a matrix or "gel".
• Most commonly, the gel is cast in the shape of a thin slab, with wells for loading the
sample.
• The gel is immersed within an electrophoresis buffer that provides ions to carry a
current and some type of buffer to maintain the pH at a relatively constant value.
• The gel itself is composed of either agarose or polyacrylamide, each of which has
attributes suitable to particular tasks.
• In electrophoresis, the charged molecules under the influence of electric field migrate
towards oppositely charged electrode.
• Those molecules with positive charge move towards cathode and negatively charged
molecules move towards anode.
• The migration is due to charge on the molecules and potential applied across the
electrodes.
PRINCIPLE

6. Factors affecting Electrophoresis
Electric field
• An electric field is a property that describes the space that surrounds electrically charged particles.
• It is defined as the electric force per unit charge.
• This electric field exerts a force on other charged objects and is radially outward from a positive charge and radially in
toward a negative point charge.
• A movement of ions depends upon voltage, current, and resistance of the electric field
Voltage
• Travel time of the molecules being separated is affected by the voltage applied.
• The higher the voltage, the faster DNA will travel through the gel.
• Voltages that are too high can possibly melt the gel or cause smearing or distortion of DNA bands.
• If the separation of the electrodes is d (meters) and the potential difference between them is V (volts), the potential
gradient is V/d volts m-1.
• The equation is Vq/d newtons, if force on the ion with a charge is q (coulombs).
• The rate of migration is proportional to Vq/d, so it increases with increase in potential difference.
• Voltage is the potential energy of electrical supply stored in the form of electrical charge.

7. Current
• Current is generated due to potential difference applied between the electrodes.
• It is a continuous and uniform flow of electrons around a circuit that are being pushed by the voltage
source.
• Current is measured in coulombs sec-1.
• The current is mainly conducted between the electrodes by buffer ions.
• Thus, increase in voltage will increase total number of charge towards the electrode.
• The distance traveled by the ions is directly proportional to the current and the time.
Factors affecting Electrophoresis

8. 2. The Sample
• Charge, size and shape of the sample being separated affect its own migration rate.
• A net increase in the charge increases the rate of migration.
• In accordance with Henderson-Hasselbalch equation, magnitude of charge is pH-dependent.
• Rate of migration is affected by increase in size of molecule (inversely proportional) and difference in shape
of the sample.
3. The Buffer
• Buffer affects migration rate of a compound and stabilizes the pH of the supporting medium.
• It has been observed that zwitterionic buffers are able to withstand prolonged electrolysis much better in
comparison to the traditional buffers especially in capillary zone electrophoresis.
Composition
• Most commonly used buffers for the electrophoresis are formate, EDTA, pyridine, Tris, barbitone acetate, and
citrate.
• The buffer should never bind to the molecules being separated as it effects the migration of the sample.
• In its simplest form, a buffered solution contains a mixture of a weak acid and its conjugate base.
Factors affecting Electrophoresis

9. 4. The Supporting Medium
• Migration rate of compounds depends upon type of supporting medium. Inert medium is always preferred.
• The medium might cause adsorption, molecular sieving, and electro-osmosis processes that affect the electrophoretic rate.
• Adsorption causes tailing of the sample, leading to movement of sample in the form of comet rather than a band.
This reduces rate as well as resolution of the separation.
• Molecular sieving is affected by type of gel used.
• Electro-osmosis depends upon the relative charge produced between water molecules in buffer and surface of supporting
material.
Factors affecting Electrophoresis

10. Factors affecting Electrophoresis Electrophoretic mobility depends on :
■ Charge: higher the charge greater the electrophoretic mobility.
■ Size: bigger the molecule, greater are the frictional and electrostatic forces
exerted on it by the medium. Consequently, larger particles have smaller
electrophoretic mobility compared to smaller particles.
■ Shape: rounded contours elicit lesser frictional and electrostatic retardation
compared to sharp contours. Therefore, globular proteins move faster than
fibrous ones.

11. Factors influencing Electrophoresis
Movement of proteins depends on various aspects.
Within the gel the molecules must pass through as they are moving from one pole to another.
The smaller molecules can weave in and out of the matrix of the gel with more ease, compared with larger molecules.
As a general rule, the molecules move rapid if it has more net charge, has a shape of ball and shorter diameter
1) The buffer pH
• It will influence the direction and rapid of the protein migration.
• Movement of proteins depends on various aspects; one of them is the charges on the proteins.
• Proteins are sequence of amino acids that can be ionized depend on their acid or basic character.
• The protein’s net electric charge is the sum of the electric charges found on the surface of the molecule as a function of
the environment.
• The rate of migration will depend on the strength of their net surface charges: The protein that carries more +ve charges
will move towards the cathode at a faster rate.
• On the contrary, the protein that carries more -ve charges will move towards the anode at a faster rate.
• Proteins can be separated based on their electric charges.
• Depending on the pH of the buffer, proteins in a sample will carry different charges.

12. • At the pI (isoelectric point) of a specific protein, the protein molecule carries no net charge and does not migrate in
an electric field.
• At pH above the pI, the protein has a net negative charge and migrates towards the anode.
• At pH below the pI the protein obtains a net positive charge on its surface and migrates towards the cathode.
2) The buffer ionic strength
• It influences the proportion of the current carried by the proteins.
• At low ionic strength the proteins will carry a relatively large proportion of the current and so will have a
relatively fast migration.
• At high ionic strength, most of the current will be carried by the buffer ions and so the proteins will migrate
relatively slowly.
• In electrophoresis, therefore, a low ionic strength is preferred as it increases the rate of migration of proteins.
• A low ionic strength is also preferred as it gives a lower heat generation.
• Assuming a constant voltage, if the ionic strength is increased, the electrical resistance decreases but the current
will increase.
• A high ionic strength buffer will therefore lead to greater heat generation, and so a low ionic strength is
preferred.
Factors influencing Electrophoresis

13. 3) The voltage gradient
The rate of migration will depend on the voltage gradient: There is more voltage gradient in the electric field, protein
will move towards the anode (or the cathode) at a faster rate.
4) Electo-osmosis
• Liquid’s relatively move upon solid medium in an electric field is called electo-osmosis.
• In applied electric field, electo-osmosis distorts the sample stream and limits the separation.
• Paper electrophoresis has poor resolution because of electo-osmosis.
• The surface of paper has -e, so the buffer has +e derived from hydrogen ions because of electrostatic induction.
• Then +e drive buffer to cathode in electric field, these flows distort the electrophoretic migration of sample by
causing a varying residence time.
• Thus, sample will move more or less than normal.
Factors influencing Electrophoresis

14. ADVANTAGES:
Useful in biochemical investigations.
Small quantity of sample can be analyzed.
Low Cost and easy maintenance.
DISADVANTAGES:
Unsuitable for accurate mobility and isoelectric point determination.
Due to the presence of supporting medium, technical complications
such as capillary flow, electro osmosis, adsorption and molecular sieving
are introduced.
Saturation of the medium with the buffer.
Sample application.
Electrophoretic separation.
Removal of the supporting media

15. TYPES OF ELECTROPHORES
1.Capillary electrophoresis
1.Gel electrophoresis
2.Paper electrophoresis
2.Slab electrophoresis
1.Zone electrophoresis
2.Immunoelectrophoresis
3.Isoelectrofocusing