Study of

electrophoresis
Dr. Bishesh Sah
Department of Biochemistry
Nepalgunj Medical College
Study of electrophoresis
• Definition
• Principle
• Types of electrophoresis
• Clinical diagnosis
Definition
• Electrophoresis is a comprehensive term that refers to the migration
of charged solutes or particles in a medium under applied electric
field.
Analytes separated by electrophoresis
• Proteins and peptides
• Nucleotides and nucleosides
• Organic acids
• Hemoglobin variants
• Lipoproteins
• Isoenzymes, etc.
Principle of electrophoresis
• Basic principle of electrophoresis is based on charge to mass ratio.

• Depending on the charge, the particles carry, ionized solutes move

toward opposite electrode.

• Isoelectric point pI (IEP) is the pH at which a particular molecule

carries no net electrical charge.

• In a more alkaline solution (above isoelectric point), the ampholyte

becomes negatively charged and migrates toward the anode. In acidic
solution (below isoelectric point), ampholyte becomes positively
charged and migrate toward cathode.
• Proteins contain many ionizable amino (-NH2) and carboxyl (-COOH)
groups. So, they both behave as ampholyte in solution.

• The separated compounds form distinct zones or bands, which can be

detected by conventional methods like staining, autoradiography, UV
absorption or enzymatic color reactions.
1. Buffer chamber 2. Connecting wire to the power source 3. Support media
4. Wicks dipping into the buffer (for completing the electrical circuit) 5. Lid

Fig: Component of electerophoreis

Factors influencing electrophoresis
1. Supporting medium - gel is preferred

2. Buffer - optimum ionic strength of 0.05-0.1 M and alkaline pH.

3. Electric current - Electrophoretic mobility is directly proportional to the

applied voltage (volts) and current (ampere) and inversely proportional to
the resistance.

4. Size and shape of sample – Smaller and spherical have great mobility

5. Electroendosmosis: Charge of supporting medium and buffer encounter the

normal flow of sample ions which cause slow migration of ions sample.
Types of electrophoresis
1. Capillary electrophoresis
2. Zone electrophoresis
i. Paper electrophoresis
ii. Cellulose acetate elctrophoresis
iii. Gel electrophoresis
Agarose gel electrophoresis
PAGE (polyacrylamide gel electrophoresis)
SDS-PAGE (sodium dodecyl sulfate-Polyacrylamide Gel Electrophoresis)
3. Isoelectric focusing
4. Immunoelectrophoresis

Paper and gel electrophoresis are most commonly used.

Note
• If the supporting medium is a sheet of Whatman filter the technique
is called as paper electrophoresis.

• If the medium is a strip of cellulose acetate the technique is called as

cellulose acetate electrophoresis.

• If the medium is a slab of porous gel it is referred to as gel

electrophoresis agar gel, agarose gel, starch gel, polyacrylamide gel.
Fig: Polyacrylamide gel electrophoresis
General procedure
• Preparation of slides
• Loading of sample
• Electrophoretic run
• Fixing and staining
• Quantification
Applications
I. Paper electrophoresis
Separation of protein, amino acids and nucleotides.

II. Agarose gel electrophoresis

for determination of molecular weight of DNA
for DNA sequencing
to study the purity of DNA
to analyze recombinant DNA molecule.

II. PAGE
To separate enzymes and isoenzymes.
III. Applications of SDS-PAGE
To determine molecular weight of proteins.
To fractionate protein subunits.
To assess the purity of protein samples.

IV. Immunoelectrophoresis
Detection and characterization of Ag

V. Isoelectric focusing
Phenotyping variants of hemoglobin.
Separation of plasma protein in electrophoresis

• This is the most commonly employed analytical technique for the

separation of plasma (serum) proteins.

• In agar gel electrophoresis, plasma proteins is separated into 5 distinct

bands namely albumin, alpha-1, alpha-2, beta- and gamma globulins

• The concentration of each one of these fractions can be estimated by

a densitometer.

• Albumin has the maximum mobility and gamma globulin has the
minimum mobility in the electric field.
Separation of lipoprotein by electrophoresis

• The serum is applied on cellulose acetate

• Electric current is applied

• The strip is dried and stained with lipid dyes such as Oil Red O.

• As a general rule, those with higher protein content will move faster
towards the anode and those with less proteins have minimum
mobility.
Cause for abnormal pattern in
electrophoresis
1. sickle-cell anemia
• In case of sickle-cell trait, the fast moving HbA and slow moving HbS
are observed. The slow mobility of HbS is due to less negative charge
(valine), caused by the absence of glutamate residues that carry
negative charge.
2. Multiple myloma:
• Monoclonal band (M-band)
between beta and gamma.

• Monoclonal gammopathy
3. Nephrotic syndrome – Decreased albumin with
sharp and prominent alpha-2 macroglobulin.
4. Primary immune deficiency:
• The gamma globulin fraction is reduced.

5. Cirrhosis of liver :
• Albumin synthesis by liver is decreased,
with a compensatory excess synthesis of
globulins by reticuloendothelial system.
• albumin band will be thin, with a wide
beta fraction;
• sometimes beta and gamma fractions
are fused (due to increased IgA).
Fig: Liver cirrhosis
6. Chronic lymphatic leukemia : gamma globulin fraction is reduced.

7. Alpha-1 antitrypsin deficiency : The alpha-1 band is thin or even missing.

8. LDH patterns in electrophoresis

1 2 3 4 5

Normal Normal