basic introduction to electrophoresis

1. ELECTROPHORESIS
Dipesh Tamrakar
M.Sc. Clinical Biochemistry
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2. OVERVIEW
 Introduction
 History
 Principle
 Factors affecting electrophoresis
 Instrumentation
 Basic procedures
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3. INTRODUCTION
• Electrophoresis is the movement of charged particles through an
electrolyte when subjected to an electric Field at the given pH
• Iontophoresis refers to the migration of small ions
• zone electrophoresis is the migration of charged macromolecules in
a porous support medium such as paper, cellulose acetate, or
agarose gel film.
• Cations move towards cathode and Anions move towards anode
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4. • By this technique solutes are separated by their different rates of
travel through an electric field.
• In a clinical laboratory, the macromolecules of interest are proteins
in serum, urine, cerebrospinal fluid (CSF), and other biologic body
fluids and erythrocytes and tissue.
• particularly in the separations of proteins, peptides and nucleic
acids
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5. HISTORY
• Father of electrophoresis- ArneTiselius (Sweeden,1902-1971) Nobel
prize in 1948 for chemistry “for his research on electrophoresis and
adsorption analysis ,especially for his discoveries concerning
complex nature of serum protein”
• Arne Tiselius in the 1930s devised moving boundary method;
"Tiselius apparatus"
• In 1937, Tiselius with support from the Rockefeller Foundation,
developed the moving boundary electrophoresis
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6. • In the 1940s and 1950s ,Zone electrophoresis methods became
effective which used filter paper or gels as supporting media.
• By the 1960s, gel electrophoresis methods are introduced which
can separate biological molecules based on minute physical and
chemical differences.
• Gel electrophoresis and related techniques became the basis for a
wide range of biochemical methods, such as protein fingerprinting,
Southern blot, western blotting procedures, DNA sequencing, and
many more
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7. PRINCIPLE
• Depending on the kind of charge they carry, ionized solutes move
toward either the cathode (-ve) or the anode (+ve) in an
electrophoresis system.
• An ampholyte becomes positively charged in a solution more acidic
than its isoelectric point (Pi) and migrates towards the cathode.
• In a more alkaline solution it becomes negatively charged and
migrates towards anode.
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8. ELECTROPHORETIC MOBILITY
• When a potential difference (voltage) is applied across the
electrodes, it generates a potential gradient (E)= applied voltage/
distance between the electrode
• When this E is applied, the force on a molecule bearing charge of
“q” coulombs is “Eq” newtons
• It is that force that drives a charged molecules towards an
electrode
• Electrophoretic mobility () = Velocity of ion (V)/ field strength (E)
• On applying potential difference, molecules will separate
depending upon 
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9. FACTORS AFFECTING ELECTROPHORESIS
The rate of migration of a solute in an electric field depends on the
following factors-
1. Net charge on the particle
2. size and shape of the particles
3. pH of the medium
4. Strength of electric field
5. Chemical and physical properties of supporting medium
6. Electrophoretic temperature
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10. FACTORS AFFECTING ELECTROPHORESIS
• Electrophoresis velocity depends on-
• Magnitude of its charge
• Charge density
• Molecular weight
• Shape
Inherent Factors
• Solution pH
• Electric field
• Solution viscosity
• Temperature
External
environment-
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11. • Frictional resistance that retards the movement of this charged
molecules
• This frictional force is a measure of hydrodynamic size & shape of
the molecule, pore size of medium and viscosity of buffer
• The velocity ‘V’ of a charged molecule in an electric field is given by
V = Eq / f f= frictional coefficient
• Even molecules with similar charge will begin to separate if they
have different molecular size ;due to different frictional forces
• Incomplete form of electrolysis; termination of electric field before
the molecules reach electrodes
• The separated samples are then located by staining with an
appropriate dye or by autoradiography; if sample is radiolabeled
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12. • The current in the solution between the electrodes is conducted
mainly by the buffer ions, a small proportion being conducted by
the sample ions
• Ohm’s law: Resistance = voltage / current
• The distance migrated by the ions will be proportional to both
current & time
• During electrophoresis the power (Watt-W) generated in the
supporting medium is W= I2R
• Most of this power generated is dissipated as heat
• If voltage is constant & current causes  Resistance
• current causes  heat so use of stabilized power supply & low
current
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13. Heating of the electrophoretic medium has the following effects:
• An increased rate of diffusion of sample and buffer ions leading to
broadening of the separated samples.
• The formation of convection currents, which leads to mixing of
separated samples.
• Thermal instability of samples that are rather sensitive to heat. This
may include denaturation of proteins (and thus the loss of enzyme
activity).
• A decrease of buffer viscosity, and hence a reduction in the
resistance of the medium.
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14. • The phenomenon of ELECTROENDOSMOSIS affects electrophoretic
separation
• It is due to presence of charge groups on the surface of the support
medium, Examples:
–Paper has some carboxyl groups present
–Agarose contain sulphate groups
–Surface of glass walls used in capillary electrophoresis contains
silano (Si-OH ) groups
• In fused-silica capillary tube, above pH value of about 3, silanol
groups on the silica capillary wall will ionize generating negative
charge sites
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15. • These charge will generate electroendomosis
• The ionized silanol groups create an electrical double layers
• When voltage is applied, cations in the electrolyte near the
capillary wall migrate towards the cathode, pulling electrolyte
solution with them
• This creates a net electrosmotic flow towards the cathode
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16. INSTRUMENTATION
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18. 5 components of electrophoresis
1. The driving force (electrical power)
2. The buffer tank  hold the buffer
3. The support medium  separation takes place
4. The Electrodes- made of platinum or carbon  connects the
buffer to power supply.
5. The detecting system
• The electrophoresis support on which separation takes place may
contact the buffer directly or by means of wicks
• The entire apparatus is covered to minimize separation
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19. 1. POWER SUPPLIES
• Power supplies are available commercially
• The migration rate can be kept constant by using a power supply
with constant current. This is true because, as electrophoresis
progresses, a decrease in resistance as a result of heat produced
also decreases the voltage.
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20.  Electrophoresis is always carried out in an appropriate buffer.
 It is essential to maintain a constant state of ionization of the
molecules being separated.
 Buffer ions actually have a double purpose in electrophoresis:
1. They carry the applied current
2. They set the pH at which electrophoresis is carried out
• Choice of buffer depends on the nature of substance to be
separated
2. BUFFER SYSTEM
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21. • During electrophoresis, ions cluster around a migrating particle.
• The higher the ionic concentration, the higher the size of the ionic
cloud and the lower the mobility of the particle.
• Greater ionic strength produces sharper protein-band separation
but leads to increased heat production.
• This may cause denaturation of heat-labile proteins. Consequently,
the optimal buffer concentration should be determined for any
electrophoretic system.
• Generally, the most widely used buffers are made of monovalent
ions because their ionic strength and molality are equal.
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22. Considerations:
• Buffer used in electrophoresis are good culture media for the
growth of microorganism, so they should be refrigerated when not
in use.
• In addition cold buffer is preferred in an electrophoretic run
because it improves resolution and decreases evaporation from
the electrophoretic support.
• Buffer used in small volume apparatus should be discarded after
each run because of pH changes due to water electrolysis.
• Large volume of buffer used (>100ml) can be mixed from both
reservoir and can be reused.
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23. 3. SUPPORT MEDIA
• The pioneering work on protein electrophoresis by Arne Tiselius was
performed in free solution.
• Persists various problems ; adverse effects of diffusion and
convection currents, could be minimized by stabilizing the medium.
• The support medium cuts down convection currents and diffusion so
that the separated components remain as sharp zones.
A. Filter paper
B. Starch gel
C. Cellulose acetate
D. Agarose
E. Polyacrylamide gel
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24. A. FILTER PAPER
• Most appropriate support media
• Frequently used in separating small particles s/a carbohydrate,
amino acids and oligopeptides
• Not used nowadays because the polar nature of cellulose shows
absorptive effect causing diffusion of samples and broadening of
separated zones
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25. • Starch gel electrophoresis separates proteins on the basis of surface
charge and molecular size, as does polyacrylamide gel.
• The procedure is not widely used because of technical difficulty in
preparing the gel.
• Was the first gel medium to be used for electrophoresis.
• It separates proteins by both charge to mass ratio and molecular size
• Here proteins compacted on the surface of the gel before migrating
into it, they formed narrow bands with improved resolution.
B. STARCH GEL
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26. • Paper electrophoresis use has been replaced by cellulose acetate or
agarose gel in clinical laboratories.
• Cellulose is acetylated to form cellulose acetate by treating it with
acetic anhydride.
• Cellulose acetate, a dry, brittle film composed of about 80% air
space, is produced commercially.
• Cellulose acetate prepared to reduce electroendosmosis is available
commercially.
• Cellulose acetate is also used in isoelectric focusing.
• Widely used for separation of lipo-proteins, isoenzymes and
hemoglobin variants
C. CELLULOSE ACETATE
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27. • widely used supporting medium
• Linear polysaccharide of repeating unit agarbiose
• Used as a purified fraction of agar, it is neutral and, therefore, does
not produce electroendosmosis
• usually used at concentrations of between 1% and 3%.
• The dried gel can be stored indefinitely.
• requires small amounts of sample (approximately 2 mL); it does not
bind protein and, therefore, migration is not affected.
D. AGAROSE GEL
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28. • Polyacrylamide gel electrophoresis involves separation of protein on
the basis of charge and molecular size.
• Layers of gel with different pore sizes are used.
• The gel is prepared before electrophoresis in a tube-shaped
electrophoresis cell.
• Polyacrylamide gel electrophoresis separates serum proteins into 20
or more fractions rather than the usual 5 fractions separated by
cellulose acetate or agarose.
• It is widely used to study individual proteins (e.g., isoenzymes).
E. POLYACRYLAMIDE GEL
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29. METHOD
1. Separation
2. Detection
3. Quantification
• General procedure for Cellulose Acetate Paper Electrophoresis
1. Set up electrophoresis set, buffer and require stain and destain
reagents
2. Soak paper in buffer and blot additional buffer
3. Apply sample & control by applicator & transfer paper into bridge
4. Run in electrophoresis for required time at specified current,
voltage or power
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30. 5. Paper is then removed and stained, destained and cleared as per
required time
6. Detection, analysis and quantification using scan densitometer
• Most commonly used general protein detecting stain is sulphated
trimethylamine dye , Coomassie Brillant Blue- mainly for PAGE
• Dye that doesn’t stain support media: Ponceau S, Procion blue,
Amido black
• Silver stain (100 times more sensitive than CBB & detects protein
down to 0.1 ng)
• PAS stain for glycoprotein; lectins
• Quantitative analysis by scanning densitometry
• Recently bench top system UV detection
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32. #TECHNICAL AND PRACTICAL CONSIDERATION:
1. Sampling:
• To achieve a proper balance between sensitive measurements and
resolution, the amount of serum protein applied to an
electrophoretic support must be optimum.
• Urine specimens require 50-100 fold concentration or extended
application time for adequate sensitivity
• CSF may or may not require concentration, depending on the
staining approach used
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33. 2. Discontinuities in sample application
May be caused by:
a. Dirty applicators
b. Uneven absorbance by sample combs
c. Inclusion of an air bubble if sample is pipetted onto the gel.
3. Unequal Migration Rates:
Caused by:
a. Dirty electrodes causing uneven application of the electric field
b. Uneven wetting of the wicks
c. Improper placement of gel causing sagging and uneven thickness
d. Storing gels too close to heat sources causing partially and
unevenly dried areas
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34. 4. Distorted, Unusual or atypical Bands:
Distortion may be caused by:
• Bent applicators,
• Incorporation of an air bubble during sample application,
• Overapplication or inadequate blotting of the sample.
• Excessive drying of the support medium.
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35. 5. Unusual bands:
• Hemolyzed samples are frequent cause of increased beta
globulin or an unusual band between the alpha 2 and beta.
6. Atypical Bands:
• Irregular, sharp protein zone is seen at the starting point due to
denatured protein.
# Therefore , control serum should be run with each
electrophoresis which helps in evaluation of its quality.
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