Biochemistry Seminar-
Electrophoresis
Roll No.- 106 to 126
Learning Objectives
• About the process of electrophoresis
• Various types of electrophoresis
• Special emphasis on PAGE
• Applied Clinical aspect of Electrophoresis
Electrophoresis
• Movement of charged particles
through Electrolyte
• Particles subjected to an external
electric field
• Positively charged particles migrate
to the Cathode
• Negatively charged particles migrate
to the Anode
History of Electrophoresis
• Early Observations (1807):
Ferdinand Reuss, from Moscow
State University, first observed the
electrokinetic phenomenon, where
clay particles dispersed in water
migrated under an electric field.
• Tiselius's Apparatus (1930s): Arne
Tiselius developed the "Tiselius
Apparatus" for moving boundary
electrophoresis, a crucial step in
separating protein mixtures.
History of Electrophoresis
• Zone Electrophoresis (1950s):
Tiselius dubbed these methods
"zone electrophoresis". Oliver
Smithies introduced starch gel as an
electrophoretic substrate in 1955,
further expanding the applications
of zone electrophoresis in
biochemistry.
• Development of Gel Electrophoresis:
The process was initially run in fully
liquid conditions, but modifications
to run on solid matrices (gels)
allowed for the creation of discrete
bands, leading to the development
of "zonal electrophoresis".
History of Electrophoresis
• Agarose and Agar (1960s): Initially, agar,
a natural carbohydrate, was used as a
separation medium for electrophoresis,
but this was replaced in the late 1960s by
agarose, a polysaccharide which is one of
the main components of agar.
• Slab Gels (1970s): The advent of "slab"
gel formats around 1970 led to major
developments in gel technology, allowing
for simpler preparation and running
multiple samples simultaneously.
• SDS-PAGE (1970s): Ulrich Laemmli
developed SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) in 1970, a
technique widely used for protein
separation.
Factors Affecting Electrophoresis
• Net Electric charge on Molecule
• Size and Shape of the Molecule
• Ionic Strength and pH of buffers
• Strength of External Electric field
• Temperature
• Nature of supporting medium
Electrophoresis Apparatus
• Process carried out in a
large tank
• Should be suitable for
supporting medium
• Optimum pH is to be
maintained.
• Suitable medium and
buffers are chosen
• E.g.-Serum protein
separation: pH-8.6;
barbitone buffer
Electrophoresis Instrumentation
Parts of Electrophoresis Tank
• Buffer Chambers
• Electrodes
• Electrical Supply and power supply
• Region of Supporting Medium
Procedure
Support Medium for Electrophoresis
• Filter Paper
• Cellulose Acetate Membrane
• Agar or Agarose
Filter Paper
• Electrophoresis carried for 16-18 hours, low voltage
• Advantages:
• Simple medium, cost effective
• Disadvantages:
• Long Duration, blurring of margins
Cellulose Acetate Membrane
• Preferred solid support media for horizontal electrophoresis
• Advantages:
• Fast process(within one hour), Excellent separation without diffusion
• Disadvantages:
• Expensive process
• Widely used for separation and identifying:
• Lipoproteins, Isoenzymes, Hemoglobin
Agar or Agarose
• Heterogenous polysaccharides
• Viscous liquid when hot, solidify
to gel on cooling
• Process takes about 90 minutes
• Cheaper than cellulose acetate
• Commonly used to study:
Serum proteins and nucleic
acids
• Also used in
immunoelectrophoresis
Types of Electrophoresis
• Polyacrylamide Gel Electrophoresis
• Immuno-electrophoresis
• High Voltage Electrophoresis
• Capillary Electrophoresis
• Isoelectric Focusing
• Pulsed Field Gel Electrophoresis
• Two-Dimensional Electrophoresis
Immunoelectrophoresis
• Combination of Immunology and Electrophoresis
• Electrophoresis bands contain antigen
• Subjected to a parallel trough of antibodies
• Antigen-antibody reaction takes place
• Precipitin bands obtained
High Voltage Electrophoresis
• Usual Electrophoresis carried at current <250 volts
• Separation of molecules directly depends on strength of
current
• High voltage current applied(400-2,000 volts)
• Results obtained within half an hour
• Used widely for separating proteins as well as
nucleotides
Capillary Electrophoresis
• Gel is taken in a capillary tube
• Dimensions: small bore(50-100 microns); length(100-
200 cm); Volume of sample(Nanolitres); Voltage(25,000
volt)
• Apparatus connected to buffer
• Result obtained in few minutes
• Used in analysis of: Drugs, Amino acids, Proteins,
Vitamins, Carbohydrates and Nucleotides.
• Forms basis of Microchip Electrophoresis
Isoelectric Focusing
• Based on Principle: immobilization of particles at
isoelectric pH
• Stable pH gradients set up
• Isoelectric pH points of all components covered
• Sharp stationary bonds are formed at isoelectric pH
• As a result, components and immobilized in bands
• Up to 40 gel bands can be formed
• Gel bands can be stained and identified
Pulsed Field Gel Electrophoresis
• Conventional Electrophoresis- Direct current supply
• PFGE- Alternating current supply
• Direction of current alternated at regular intervals
• Alternate power supply by 2 different arrays of
electrodes
• Used for separating large and small sized particles
• Appropriate pore size gels used
• E.g.- 50kBP DNA separated from 400kBP DNA
Two-Dimensional Electrophoresis
• Electrophoresis performed twice in mutually
perpendicular directions
• First separation in based on charge
• Second Separation based on molecular size
• Used to study differences in protein content in Genetic
Disorders
• Detection of protein fragment by Autoradiography or
Coomassie blue stain
Polyacrylamide Gel Electrophoresis
(PAGE)
• High molecular sieving
effect, Separation is very
efficient
• Agarose gel can separate
only up to 5 bands
• PAGE- serum separated in
more than 20 bands
• Better control over cross
linking and pore size
Advantages & Limitations of PAGE
Advantages:
• High resolution for protein separation.
• Can separate small differences in molecular weight.
• Used in many research and clinical applications.
Limitations:
• Requires expertise and specialized equipment.
• Time-consuming compared to other separation methods.
• Staining and visualization require additional steps.
SDS-PAGE
• Common variant of PAGE
• SDS-Sodium Dodecyl Sulfate- Denaturizing Agent
• Proteins boiled with SDS for 1-2 minutes
• SDS covers negative charge on proteins
• Strang negative charge created
• Separation mainly depended on molecular size
• Commonly used to assess purity of proteins
Visualizing of Protein Bands
Steps:
• Allow Electrophoretic process to complete
• Fix protein to solid support
• Stain with dye
• Destain by dilute acetic acid
• Scanning by Densitometer
Visualizing of Protein Bands
• Possible fixatives: Methanol, Acetone
• Probable dyes: Amido Schwartz, Naphthalene black,
Ponceau S, Coomassie Blue.
• Principle of densitometer:
When light passed through Agar Gel plate, absorption
of light will be proportional to quantity of Protein present
on a band
Clinical Applications
• Analysis of Hemoglobin (e.g.- thalassemia, sickle cell
anaemia)
• Identification of M-band in Multiple myelomas
• Quantification of Lipoproteins in hyperlipoproteinemia
• Isoenzyme analysis
Sickle Cell Anemia
• HbS contains 2 normal α-globin
chains and 2 mutant β-globin chains
(βS),
• Glu at 6th position is replaced with
Val
• Electrophoresis-alkaline pH,
• HbS migrates more slowly toward
the anode than does HbA
• Altered mobility of HbS is a result of
the absence of the negatively
charged glutamate residues in the
two β chains,
• HbS less negative than HbA.
Isoenzymes
• Isoenzymes- catalyse same chemical reaction
• Have characteristic Physical and Biochemical properties
• Electrophoresis is a common technique used to
separate and identify isoenzymes
• Differences in charge, size, or isoelectric point.
• Other methods include: chromatography,
immunoassays, and mass spectrometry.
IMMUNOGLOBULINS IN MULTIPLE
MYELOMA
• Specialized proteins-defense against the foreign
substances.
• Five Classes – IgM, IgG, IgA, IgD, IgE
• In Multiple myeloma-increased production of one Clone
• Enormous proliferation of clone
• Monoclonal band spike in Electrophoresis
• IgG- most common
Vote of Thanks
• We sincerely thank the Department of Biochemistry,
Seth GS Medical College, to present us with this
opportunity
to understand Electrophoresis and its applications,
and to improve our presentation skills.
Bibliography
• Textbook of Biochemistry, 10th Edition, by DM
Vasudevan
• Biochemistry by U. Satyanarayana
• Lippincott Biochemistry
• Harper’s illustrated Biochemistry, 28th Edition
