This document discusses electrophoresis, which is the movement of charged particles in an electric field. It separates molecules based on their charge and size. Key factors that affect migration rate are listed. The main requirements for electrophoresis are an electrophoresis tank, electrodes, power supply, buffer, and specimens like serum or plasma. Common electrophoresis techniques described include zone electrophoresis using paper or gel, isoelectric focusing, immuno electrophoresis, and SDS-PAGE which separates based on size. Clinical applications involve using electrophoresis to analyze conditions like liver disease or infections.

1. TO
STUDY
ELECTROPHORESIS
BY: Kamlesh Yadav
(Msc.Medical Biochemistry)

2. DEFINITION
• Movement of charged particles (ions) in an
electric field resulting in their migration
towards the oppositely charged electrode is
called electrophoresis.
• It is widely used analytical technique for the
separation of biological molecules such as
plasma proteins, lipoproteins.

3. PRINCIPLE
• Charged molecules migrate either to cathode
or anode depending upon the kind of charge
they carry. Molecules with a net positive
charge (cations) move towards cathode
whereas molecules with net negative charge
(anions) migrate towards anode.

4. FACTORS AFFECTING THE RATE
OF MIGRATION
• Total net charge on molecules.
• Size and shape of particles.
• pH of buffer solution.
• Strength of electric field applied.
• Property of supporting medium.
• Temperature

5. REQUIREMENTS FOR ELECTROPHORESIS
• Electrophoresis tank (to hold buffer).
• Electrodes
• Power pack to supply electricity at constant
current & voltage. Power supply vary based on
type of electrophoresis and type of gel used.
(PAGE usually uses higher voltage while
Agarose gel electrophoresis generally uses
lower voltage).

6. Continued...
• Buffer
To create pH mostly around 8.6. At this pH all
serum proteins will have a net negative charge
& will migrate towards anode to conduct the
current.
• Specimen
(Serum, Plasma, Urine or Fluid CSF, Pleural
fluid).

7. TYPES OF ELECTROPHORESIS
The most commonly employed electrophoretic
techniques in lab include the following.
• Zone electrophoresis (Paper & Gel)
• Isoelectric focussing
• Immuno electrophoresis

8. MOVING BOUNDARY ELECTROPHORESIS
• Originally developed moving boundary
electrophoresis by Tiselius (1937) is less
frequently used these days.
• In this technique U- shaped tube is filled with
protein solution overlaid by a buffer solution.
• As the proteins move in solution during
electrophoresis, they form boundaries which
can be identified by their refraction index.

9. ZONE ELECTROPHORESIS
• A simple & modified method of moving
boundary electrophoresis.
• An inert supporting medium such as paper or
gel are used.
1. Paper Electrophoresis
2. Gel Electrophoresis

10. PAPER ELECTROPHORESIS
• Sample is applied on a strip of filter paper wetted
with desired buffer solution.
• The ends of the strip are dipped into buffer
reservoirs in which electrodes are placed.
• The electric currents is applied allowing
molecules to migrate for sufficient time.
• The paper called electrophoretogram is removed
dried & stained with a dye that specifically
colours the substance to be detected.
• Serum proteins are separated into separate distinct
bands of albumin, alpha 1 globulin, alpha 2
globulins, β-globulins & γ-globulins.

11. GEL ELECTROPHORESIS
• Electrophoresis that involves the use of a gelatinous
material as agarose, acrylamide, starch or cellulose
acetate as the matrix (support medium).
• It involves separation of molecules based on their
size in addition to electrical charge.
• Movement of large molecules is slow.
• Resolution is much higher & thus, serum proteins
can be separated to about twenty different bands
instead of five bands on paper electrophoresis.
• Polyacrylamide gel electophoresis (PAGE) is one of
the commonly used technique named after the gel
used.

12. POLYACRYLAMIDE GEL ELECTROPHOREIS
• Electrophoresis is carried out in polyacrylamide
gel with a characteristic pore size.
• Proteins are separated on the basis of their
charge and molecular size.
• In this technique, serum sample is applied at
the top of gel, and proteins, following
electrophoresis, are stained with amido black.
• It may yield 20 or more fractions.
• widely used to study individual proteins in
serum, genetic variants and isoenzymes.

13. • PAGE can also be carried out in the presence
of sodiumdodecyl sulphate (SDS), called as
SDS-PAGE.

14. SDS-PAGE
• Stands for sodiumdodecyl sulphate-
polyacrylamide gel electrophoresis.
• Variant of PAGE.
• Proteins are boiled for one or two minutes with
SDS which is a denaturing agent.
• The negative charges of SDS cover the protein
molecules making them strong negative.
• Then the separation will depend mainly on
molecular size.
• It is commonly used for molecules weight
determination as well as for assessing the purity
of proteins.

15. ISOELECTRIC FOCUSSING
• Primary based on immobilization of molecule
at iso-electric pH during electrophoresis.
• Serum proteins can be separated to as many as
40 bands.
• Can be conveniently used for the purification
of proteins.

16. IMMUNO ELECTROPHORESIS
• It involves combination of the principles of
electrophoresis & immunological reactions.
• Used for analysis of complex mixture of
antigens & antibodies.

17. APPLICATIONS
• DNA sequencing
• Blotting
• Medical Research
• Protein Research
• Drugs
• Vitamins
• Carbohydrates
• Agricultural testings

18. CLINICAL APPLICATIONS
DISEASE
• Hepatic Cirrhosis
• Nephrotic Syndrome
• Multiple myeloma
• Protein energy malnutrition
• Acute infection
• Chronic infection
ELECTROPHORESIS
FINDINGS
• Albumin decrease/ band γ-
Globulin increases.
• Alpha-2-globulin increases
• N-band seen between β & γ
globulin.
• Albumin decreases
• Increase in α-2-globulin.
• Increase in both α-1 & α-2
globulin.

21. Thank you