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Applied Biochemistry

ELECTROPHORESIS - HEMALA DEVI S

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By: S.Hemala Devi
INTRODUCTION:
Electrophoresis is a physical method of analysis
which involves separation of the compounds that are
capable of acquiring electric charge in conducting
electrodes.
DEFINITION:
 Electrophoresis is the motion of dispersed
particles relative to a fluid under the influence of
a spatially uniform electric field.
 Electrophoresis of positively charged particles
(cations) is sometimes called cataphoresis.
Applied Biochemistry
 Electrophoresis of negatively charged particles
(anions) is sometimes called anaphoresis.
 The electrokinetic phenomenon of electrophoresis
was observed for the first time in 1807 by Russian
professors Peter Ivanovich Strakhov and Ferdinand
Fredric Reuss at Moscow State University ,who
noticed that the application of a constant electric
field caused clay particlees 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 analytical techniques used in
chemistry for separating molecules by size, charge,
or binding affinity .
 Electrophoresis is used in laboratories to separate
macromolecules based on size .
 The technique applies a negative charge so proteins
move towards a positive charge .
 Electrophoresis is used extensively in DNA ,RNA and
protein analysis .
PRINCIPLE:
 Electrophoresis is general term that involves the
migration and separation of charged ions under the
influence of electric current .
 It consists of two electrode anode and cathode and
electrolyte which serve as conducting medium .
IMPORTANCE OF ELECTROPHORESIS:
 It is used to separate macromolecules like DNA , RNA
and proteins .
 DNA fragments are separated according to their
size.

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Applied Biochemistry

  • 2. INTRODUCTION: Electrophoresis is a physical method of analysis which involves separation of the compounds that are capable of acquiring electric charge in conducting electrodes. DEFINITION:  Electrophoresis is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field.  Electrophoresis of positively charged particles (cations) is sometimes called cataphoresis.
  • 4.  Electrophoresis of negatively charged particles (anions) is sometimes called anaphoresis.  The electrokinetic phenomenon of electrophoresis was observed for the first time in 1807 by Russian professors Peter Ivanovich Strakhov and Ferdinand Fredric Reuss at Moscow State University ,who noticed that the application of a constant electric field caused clay particlees dispersed in water to migrate .  It is ultimately caused by the presence of a charged interface between the particle surface and the surrounding fluid.
  • 5.  It is the basis for analytical techniques used in chemistry for separating molecules by size, charge, or binding affinity .  Electrophoresis is used in laboratories to separate macromolecules based on size .  The technique applies a negative charge so proteins move towards a positive charge .  Electrophoresis is used extensively in DNA ,RNA and protein analysis .
  • 6. PRINCIPLE:  Electrophoresis is general term that involves the migration and separation of charged ions under the influence of electric current .  It consists of two electrode anode and cathode and electrolyte which serve as conducting medium . IMPORTANCE OF ELECTROPHORESIS:  It is used to separate macromolecules like DNA , RNA and proteins .  DNA fragments are separated according to their size.
  • 7.  Proteins can be separated according to their size and their charge. APPLICATIONS:  To study homogenecity of a macromolecular system.  Analysis of complex biological mixtures .  Separation of organic acid, alkaloids, carbohydrates, amino acids, alcohols, phenols, nucleic acids and insulin.  Estimation of the DNA molecule.
  • 8.  It is also used for the separation of vitamins.  Electrophoresis in combination with autoradiography is used to study the binding of iron to serum proteins. ADVANTAGES:  High separation efficiency .  Short analysis time .  Low sample and electrolyte consumption .  Low waste generation.  Ease of operation.
  • 9. DISADVANTAGES:  Gel preparation and casting – exacting n time consuming.  Complete reproducibility of gel preparation not possible.  Elaborate optical system are required.  Due to small diameter of the capillary tube , heat is dissipated that causes increased diffusion. Due to this the resolution is not always proper.
  • 10. FACTORS AFFECTING ELECTROPHORESIS:  Electrophoretic mobility depends on charge, size and shape.  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 .
  • 11.  Therefore, globular proteins move faster than fibrous ones.  Electrophoresis can broadly be divided two types:  Slab Electrophoresis  Capillary Electrophoresis
  • 12. SLAB ELETROPHORESIS :  Slab – gel electrophoresis is the predominant technique for the separation of peptides, proteins , and polynucleotides.  The slab - gel format provides mechanical stability for the separation , reduces solute dispersion from convection and diffusion , and permits handling for detection , scanning , storage .
  • 14.  It is primary method used in clinical chemistry lab.  It has ability to simultaneously separate several samples in one run.  It uses a rectangular gel regardless of thickness .  Gels are cast on sheets of plastic backing.  It is useful in separation of serum proteins , iso-enzymes, lipoproteins , haemoglobin and fragments of DNA and RNA.
  • 15.  The slab electrophoresis is further divided into three types based on the principle used for separation. Zone Electrophoresis Iso electric - Focusing Immune - Electrophoresis
  • 16. ZONE ELECTROPHORESIS:  Here, the charged particles are separated into different zones or band in a buffer and stabilized in solid porous or any other support medium.  Ex : Agar gel, Filter paper or Poly acryl amide gel.  This is of two types : Paper Electrophoresis Gel Electrophoresis
  • 18.  This technique is useful for the seperation of small charged molecules such as amino acids and small proteins .  A drop of sample is applied in a band to a thin sheet of supporting material , like paper , that has been soaked in a slightly – alkaline salt solution .  Paper electrophoresis employs filter paper strips soaked in buffer solution , usually diethylbarbituric acid and barbituric acid dissolved in alkali (Veronal buffer),pH 8.6 .
  • 19.  A small volume of serum is placed on the paper and a direct current passed for several hours . GEL ELECTROPHORESIS:
  • 20.  Gel electrophoresis is a method for separation and analysis of macromolecules and their fragments , based on their size and charge .  It is used in the clinical chemistry to separate proteins by charge or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length , to estimate the size of DNA and RNA fragments or to separate proteins by charge .  Nucleic acid molecules are separated by applying an electric field to move the negatively charged molecules through a matrix of agarose or other substances.
  • 21.  Shorter molecules move faster and migrate than longer ones because shorter molecules migrate more easily through the pores of the gel.  This phenomenon is called sieving .  Proteins are separated by charge in agarose because the pores of the gel are too large to sieve proteins .  Gel electrophoresis can also be used for separation of nano particles.
  • 22. ISO ELECTRIC FOCUSING:  Based primarily on immobilization of molecules at isoelectric pH during electrophoresis.  Isoelectric pH is set at different foci and hence , the molecules are immobilized at their iso – electric point .  They don’t move towards the electrodes , but stay at their iso-electric pH .
  • 23.  Very efficient to separate the proteins and from serum, almost 40 bands of proteins can be formed. Stable pH gradients are set up, usually in gel, covering the pH range to include the iso- electric points of components in a mixture . As electrophoresis occurs, molecules, migrates to positions corresponding to their iso-electric points, get immobilized and form sharp stationary bonds.
  • 24. Gel blocks can be stained and identified. Iso-electric focusing can be conveniently used for the purification of proteins. IMMUNO ELECTROPHORSIS:  Involves combination of the principles of electrophoresis and immunological reactions .  First proteins are separated on to the electrophoresis paper, then, the antibodies are allowed to diffuse through the paper and react with separated protein molecules in bands.
  • 25.  Useful for analysis of antigens and antibodies.  The antibodies diffuse and when they come in contact with antigens, precipitation occurs , resulting in the formation of precipitin bands. CAPILLARY ELECTROPHORESIS:
  • 26.  Technique first described by – Jorgensen and lukacs (1980’s) .  It is also referred as :  High performance capillary electrophoresis (HPCE)  Capillary zone electrophoresis(CZE)  Free solution capillary electrophoresis (FSCE)  Capillary gel electrophoresis(CGE)  Capillary isoeletric focusing ( CIEF )  Micellar electro kinetic chromatography(MEKC)
  • 27.  Used for size and shape separation .  Separation based on differences in solute size. It is used for protein analysis. Detection is by UV absorbance of chromohore. It is used for DNA sequencing.