Electrophoresis is a method for separating charged particles in a solution under an electric field, with applications in protein analysis and various clinical studies. It can be performed using different techniques, such as zone electrophoresis and paper electrophoresis, each having its own advantages and disadvantages. Factors affecting migration include charge, size, shape, electric field strength, and buffer composition, making it a versatile tool in biochemistry, genetics, and forensics.

1. Prepared By: Vipin Kr Shukla
Assistant Lecturer

2. Introduction:
 Electrophoresis may be defined as the migration of the charged particle
through a solution under the influence of an external electrical field.
 Ions that are suspended between two electrodes tends to travel towards
the electrodes that bears opposite charges.
 Depending on kind of charge the molecule carry, they move towards
either:
 To cathode Or to Anode
 An ampholyte become positively charged in acidic condition and migrate
to cathode, in alkaline condition they become negatively charge and
migrate to anode.

3. Electrophoresis Apparatus:

4. Contd….
 It is a type of protein separation method which relies on protein
sizes to segregate the mixture.
 It is one of the highly efficient techniques of analysis and sole
method for separation of proteins for western blot, RNA studies etc.
 But, on negative side it also time-consuming, expensive and
technical skilled procedure due to which is less preferred in health
care
 Electrophoresis is similar to other separation techniques like
chromatography but it differs in-terms of the types of samples
analyzed, the method used for separation, principle used etc.

5. ELECTROPHORESIS & ITS PRINCIPLE:
 Electrophoresis is a method of separation where in charged
molecules migrate in differential speeds in an applied electric
field.
 When electricity is applied to the medium containing biological
molecules, depending on their net charge and molecular size,
they migrate differentially, thus different proteins/DNA can be
separated.
 Depending on the kind of charge the molecule carry, they move
towards either to
 To cathode or anode.

8. FREE ELECTROPHORESIS:
 In this type of electrophoresis a free electrolyte is taken in
place of supporting media.
 It is mostly of two types:
 Micro Electrophoresis : It is mostly used in calculating Zeta
potentials(a colloidal property of cells in a liquid medium)of
the cells.
 Moving boundary Electrophoresis which for many years
had been used for quantitative analysis of complex mixtures of
macromolecules , esp. Proteins.

9. ZONE ELECTROPHOROSIS:
 It involves the migration of the charged particle on the supporting
media can be Paper, Cellulose acetate membrane, Starch Gel,
Polyacrylamide.
 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.
 ADVANTAGES:
 Useful in biochemical investigations.
 Small quantity of sample can be analyzed.
 Cost is low and easy maintenance.
 DISADVANTAGES:
 Unsuitable for accurate mobility and isoelectric point determination.
 Due to the presence of supporting medium, technical complications.

10. PAPER ELECTROPHORESIS:
 Paper Electrophoresis is one of the type of zone
electrophoresis.
 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 towards the
anode.

11. EQUIPMENTS:
 The equipment required for electrophoresis consist a basically of
two items, a POWER PACK and ELECTROPHORETIC
CELL.
 1. Power pack: Power pack provides a stabilized direct current
& has controls for both voltage & current out put, which have an
out put of 0 to 500V and 0 to 150mA are available.
 2. The Electrophoretic cell: It contains: the electrodes, buffer
reservoirs, a support for paper and a transparent insulating cover.
The electrodes are usually made of platinum.

12. WORKING:
 1) A long strip of filter paper is moistened with a suitable buffer
solution of the desired p H and the sample is applied transversely
across the central part of the strip.
 2) Ends are fixed to dip in buffer solutions in two troughs fitted
with electrodes.
 3)Electric field of about 20 volts/cm is established.
 4)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.

13. Contd….
 5)Homogeneous group of particles migrate as a separate band
 6)The electrophoresis is carried out for 16-18 hours.
 7)Proteins are stained (Bromphenol blue) to make them visible
 8) The separated proteins appear as distinct bands.

15. PROCEDURE FOR ELECTROPHORESIS

16. This ensures that the
electric current goes
through the whole tank
and that maintains
that ions can move in the
solution

17. Safety cover is put over the top and the current is switched on.
The dye will migrate through the gel toward the positive electrode, as will the DNA.
Depending on how much voltage is applied and how warm the gel is and size and shape of
molecules will depend on how fast the ions move through the gel.
Smaller fragments will move easier so they will be closer to the positive electrode.
Once the dye has moved through the gel to the buffer, the electrical current is switched off
and gel is removed from the tray.

18. VISUALISATIO
N:
 After the electrophoresis is complete, the molecules in the gel can
be stained to make them visible.
 Ethidium bromide, silver, or coomassie blue dye may be used for
this process.
 If the analyte molecules fluoresce under ultraviolet light, a
photograph can be taken of the gel under ultraviolet lighting
conditions.
 If the molecules to be separated contain radioactivity added for
visibility, an autoradiogram can be recorded of the gel.

19. FACTORS AFFECTING SEPARATION:
 The Sample- Charge- Higher the charge greater the mobility
 Size- Bigger the molecule greater the frictional and
electrostatic forces exerted on it by the medium i.e. larger
particles have smaller electrophoretic mobility compared to
smaller particles.
 Shape- The globular protein will migrate faster than the
fibrous protein.

20. Electric field:
 Increase of migration with the increase of voltage gradient.
 Buffer- Migration of charge particle depend on of the buffer.
 Composition Commonly used buffers are “Formate”,
“Acetate”, “Citrate”, “ Phosphate”, “EDTA”.
 The choice of buffer depends upon the type of sample being
electrophoresed.

21. pH:
 The extent of ionization depends on pH, especially in organic compounds.
The ionization increases with increase in pH of an organic acids and its
just reverse for the organic bases therefore affecting its rate of migration..
 The Medium :
 The inert medium can exert adsorption ,molecular sieving effects &
electro-osmosis – processes that affect the electrophoretic 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.

22. APPLICATIONS:
 Paper electrophoresis has emerged as a simple, inexpensive,
and accurate laboratory procedure for various research and
clinical studies.
 Clinical applications of paper electrophoresis include study of
sickle cell disease, hemoglobin abnormalities, and separation of
blood clotting factors and serum plasma proteins from blood
sample.

23. Contd….
 It has also been used in separation and identification of
alkaloids.
 PE can also be used for testing water samples, toxicity of
water, and other environmental components.
 Drug-testing industry uses paper electrophoresis to determine
presence of illegal drugs crime suspects.

24. Contd….
 Forensics.
 DNA fingerprint of a criminal.
 Molecular Biology To separate and organize DNA and RNA by
size.
 Genetics Provide clearer picture of DNA, it also helps prepare
DNA for cloning and genetic engineering.
 Microbiology Information out about the organisms. Virology : to
help diagnose different strains of viruses.
 Biochemistry Mapping of cellular components, particularly
proteins and nucleic acids.