Electro

UDMLS22
ELECTROPHORESIS
ELECTROPHORESIS
Electrophoresis refers to migration of charged solutes or particles in a liquid medium under the influence of an electric
field (electric current).
Widely practiced is zone electrophoresis which refers to the migration of charged macromolecules in a porous
supporting medium such as cellulose acetate paper or agarose gel film.
The end product of zone electrophoresis is an electrophoretogram

Solutes analyzed includes protein in serum, urine, csf and other biological fluids as well as protein in rbcs and tissues
PRINCIPLE OF ELECTROPHORESIS
When charged molecules are placed in an electric field, they migrate toward either the positive or negative pole
according to their charge. Electrophoresis exploits the fact that different ions have different mobility in an electric field
THEORY OF ELECTROPHORESIS
Chemical species carrying an electric charge by virtue of ionization will move either to the cathode or to
the anode in an electrophoresis system.
The kind of solutes separated are ampholyte and are said to be amphoteric in nature i.e they can either be
positively or negatively charged depending on the ph of the solution they are contained in. These solutes
have an iso-electric point (PI) i.e a point where the solute has no charge. These types of solutes can also be
referred to as zwitterions.
TYPES OF ELECTROPHORESIS
There are six types of electrophoresis namely:-
• Paper electrophoresis(PE),
• Agarose Gel Electrophoresis (AGE),
• Cellulose Acetate Electrophoesis (CAE),(4)
• Polyacrylamide Gel electrophoresis (PAGE),
• Starch Gel Electrophoresis (SGE),
• Isoelectric Focusing (IEF).
1) Paper electrophoresis(PE)
Was used to separate serum proteins but has been replaced with cellulose or agarose
Its advantages are – High tensile strength, low cost and ease of handling
Its disadvantages are – Long separation time (16 hrs) , excessive background (tailing),
decreased resolution,
(2) Agarose Gel Electrophoresis (AGE)
Has been used in analysis of serum proteins, hemoglobin variants, and lactate
dehydrogenase isoenzymes and lipoproteins fractions.
Its Advantages are; versatile, convenience
It has a low affinity for proteins hence little effect on their migration
Usually 0.5-1.0 g of agarose /dl of buffer provides a gel with the desired strength and with
good migration properties
3. Polyacrylamide Gel electrophoresis (PAGE
The previous medias yields 5-7 zones while PAGE yields 20 or more hence prefffered for studying individual serum
proteins
Separates macromolecular ions on the basis of both surface charge and molecular size.
PAGE technique employs layers of gel that differ in composition and pore size. Because of the discontinuities of the
electrophoretic matrix and the discoid shape of the separated zones of protein, this method is sometimes referred to as disc
electrophoresis.
Polyacrylamide Gel is thermal stable, transparent, strong, and relatively chemically inert
This gels are uncharged, thus eliminating electroendosmosis.
4. Starch Gel Electrophoresis (SGE)
Starch gel may be used in a horizontal process or with migration taking place in the vertical direction. Proper preparation
of gel is relatively difficult and requires considerable skill. The starch concentration is 10-16 g/dl and the ph of the buffer
varies according to the specific separation
5. Isoelectric Focusing (IEF)
The compounds to be separated (eg proteins) migrate to the zone in the medium where the ph is equal to the isoelectric
point (pI) of each compound. In that zone, the charge on the protein becomes zero and migration ceases. With IEF,
separated protein zones are very sharp because the pI of a protein is confined to a narrow pH range and because diffusion
of the protein is counteracted by acquisition of charge as it moves away from its (pI) position
PRINCIPLE OF SERUM PROTEIN ELECTROPHORESIS
Protein molecules in an alkaline medium (PH 8.6) are negatively charged and when subjected to an electric current will
migrate towards the anode (positively charged side) of the electrophoretic system. The migration will progress within a
supporting medium, for a standard period of time. The protein molecule will be separated into its various charged particles
namely: albumin, alpha 1-, alpha 2-,beta- and gamma globulins
FACTORS THAT CONTROL THE MIGRATION OF PROTEIN MOLECULES
(1) Net electrical charge of the molecule - molecules with more net electrical charge will migrate faster than the ones with less.
(2) Size and shape of the molecules - Increase in the size of protein molecule will decrease the migration .Irregular shaped
protein molecules will migrate slower than regular shaped protein molecules.
(3) Viscosity of the medium (electrophoretic buffer - migration of protein molecule will be faster in medium of low viscosity
than that of high viscosity.
(4) Strength of the electrical field - The current applied should be enough to cause the migration of the charged protein
molecules.
(5) Temperature of operation-----The temperature in the electrophoretic tank should be enough not to cause evaporation of the
buffer.
(6) Buffer properties (supporting medium properties) ---the buffer used in electrophoresis has two-fold purpose: a) they carry
the applied current, b) they fix the Ph at which the electrophoresis is carried out (ie they determine the electrical charge on the
solute).
Examples of buffers used are – Barbital buffers and Tris buffers

PROCEDURE FOR PROTEIN ELECTROPHORESIS
(a) Materials required:
• Sample (serum, urine, plasma, or CSF)
• Electrophoretic apparatus including the power pack (supply)
• Electrophoretic supporting medium (cellulose acetate paper or agarose gel)
• Buffer solution (barbital (sodium barbital) or tris buffers
• Stain eg ponseau S, methylene blue or silver nitrate
INTERPRETATION OF RESULTS
• The result of protein electrophoresis is an electrophoretogram. In a normal serum, five protein bands are expressed. The
expression from the fastest to the slowest is as follows: albumin, alpha 1 globulin, alpha 2 globulin, beta globulin and
gamma globulin.
• Densitometry is a method used for interpretation. It involves the use of a densitometer which give the location and
intensity of the zones (bands) as successive peaks on a recorder chart.
• Another method of interpretation is the ELUTION method. This involves cutting of the specific bands and eluting the
stain using suitable solvents such as basic buffers or alcoholic solutions.
Appearance of a normal electrophoretogram

LIMITATIONS AND ERRORS IN ROUTINE ELECTROPHORESIS
• Wick flow
-Heat generated causes evaporation of solvent from the electrophoretic support
-Drying effect causes buffer to rise from both tank compartments
-Flow of buffer from both directions affects protein migration
• Electroendosmosis
- Some electrophoretic support media take the negative charge due to adsorption of OH - when in contact with water
- Since the ions are fixed on the surface of the electrophoretic support they are rendered immobile relative to the other
ions in solution
- Positive ions in solution cluster about the fixed negative charge ions sites forming an ionic cloud of mostly positive
ions
- Application of electric current causes the positive ions in ionic cloud to migrate towards the negative side of the system
thus affecting the flow of the intended negatively charged solutes
- Electro endosmosis strong medium ( eg cellulose acetate paper and agarose gel ) and with minimal
- Electroendosmosis(starch, polyacrylamide gel ,highly purified agarose)
• Buffers storage
Growth of micro-organisms when stored at room temperatures and effect of electrolysis of water during electrophoresis
• Amount of stain solution
Recommended volume approximately 100ml for 387cm2 of cellulose acetate or agarose film, tightly closed to avoid
evaporation
• Sample application
Amount applied must be optimal and small enough to avoid overloading

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UDMLS22

The end product of zone electrophoresis is an electrophoretogram

PRINCIPLE OF SERUM PROTEIN ELECTROPHORESIS

Examples of buffers used are – Barbital buffers and Tris buffers

Appearance of a normal electrophoretogram

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