electrophoresis lecture

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