ELECTROPHORESIS

Dr. Ashish Agravatt

Associate Professor

Department of Biochemistry

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Electrophoresis

ELECTROPHORESIS

• Introduction
• Theory of electrophoresis
• Instrumentation
• Types
• Clinical applications

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INTRODUCTION

• It is a type of Separative Procedure widely used in clinical & research laboratories
• Used to separate complex biomolecules

like Proteins, Amino acids, Peptides,

Carbohydrates, DNA fragments, RNA etc.

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ELECTROPHORESIS:

• Electro – Refers to the energy of

electricity.

• Phoresis – From the Greek verb “Phoros”

means to “carry across”.

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DEFINITION:

• Migration of charged particles or solutes

of any size in a liquid medium under the

influence of an electric field.

HISTORY

• The first technique of electrophoresis introduced was the free solution or moving boundary method devised by ARNE TISELIUS in 1937 - FATHER OF ELECTROPHORESIS

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THEORY OF ELECTROPHORESIS

• Biological molecules such as Amino acids, proteins, peptides, nucleotides & nucleic acids possess ionizable groups.
• Depending on the kind of charge they carry, ionized solutes move toward either the cathode (negative electrode) or the anode (positive electrode) in an electrophoresis system.
• Positive ions (Cations) → Cathode
• Negative ions (Anions) → Anode

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Electrophoresis

• Molecules having similar charges move in the same direction.
• But because of their difference in the molecular mass the extent to which these they move differs
• Hence the difference in the Charge : Mass ratio (C/M) forms the basis of differential migration of particles in an applied electric field.
• And this forms the general principle of electrophoresis.

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PRINCIPLE OF SEPARATION

• According to charge
• According to Size

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ACCORDING TO CHARGE

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• When charged molecules are placed in an electric field, they migrate toward either the positive (anode) or negative (cathode) pole according to their charge.

ACCORDING TO SIZE

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Mice run faster through the forest than elephant

• Ampholyte:- A molecule that is either positively or negatively charged (zwitterion).
• Acidic medium → Positive charge → Cathode
• Alkaline medium → Negative charge → Anode

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Electrophoresis

• pH = pI 🡪 No net charge 🡪 No mobility in electric field
• pH < pI 🡪 Net positive charge (cation) 🡪 Moves towards cathode (-ve electrode)
• pH > pI 🡪 Net negative charge (anion) 🡪 Moves towards anode (+ve electrode)

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Electrophoresis

• Proteins contain many ionizable amino (-NH2 ) and carboxyl (-COOH) groups.
• Bases in nucleic acids may also be positively or negatively charged.
• Both behave as ampholytes in solution.

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Electrophoresis

• Rate of migration of ions in an electrical field depends on factors:-
• Net charge of the molecule
• Size and shape of the molecule
• Strength of the electric field
• pH of the medium
• Properties of the supporting medium
• Temperature of operation

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Electrophoresis

• The Electrophoretic mobility :-
• Directly proportional to the net charge
• Inversely proportional to the size of the molecule and the viscosity of the electrophoresis medium.

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�INSTRUMENTATION�

• Although modern electrophoresis equipment and systems vary considerably in form and degree of automation, the essential components common to all systems:-
• Power pack
• An Electrophoresis unit
• Electrophoresis units are available as :

Vertical gel systems

Horizontal gel systems

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Electrophoresis

• Vertical slab gel units
• Routinely used to separate proteins in acrylamide gels.
• Horizontal gel systems
• Two reservoirs which holds buffer
• Platinum or carbon electrodes connected to

power supply

• Support medium
• Wicks
• Cover:-To minimize evaporation

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Electrophoresis

• Power Supplies:-
• Drives the movement of ionic species in the medium and allows adjustment and control of either the current or the voltage.
• In more sophisticated units, the power may be controlled as well and conditions may be programmed to change during electrophoresis.

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• Buffers:-
• A mixture of weak acid and its base.
• Carry the applied current.
• Fix the pH at which electrophoresis is carried out.
• Thus, they determine the kind of electrical charge on the solute, the extent of ionization of the solute, and therefore the electrode toward which the solute will migrate.

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Electrophoresis

• For the separation of serum proteins, the Barbitone or Tris-boric acid-EDTA buffers remain the most popular.
• The Buffer's ionic strength determines the thickness of the ionic cloud (buffer and non buffer ions) surrounding a charged molecule, the rate of its migration, and the sharpness of the electrophoretic zones.

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Electrophoresis

• With increasing concentration of ions, the ionic cloud increases in size, and the molecule becomes more hindered in its movement.
• A relatively high ionic strength buffer containing calcium lactate has been used in High-resolution electrophoresis to improve the separation of serum proteins into as many as 13 bands.

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�SUPPORT MEDIA�

• Provides the matrix in which protein separation takes place.
• e.g. Sheets, Slabs or columns of starch, Agarose or Polyacrylamide, Cellulose acetate membrane
• Separation is based on differences in Charge-to-mass ratio of the proteins and, depending on the pore size of the medium, possibly Molecular size.

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Electrophoresis

• Starch Gel:-
• Prepared from partially hydrolyzed native starch.
• Use:- Starch gel electrophoresis (SGE) to separate macromolecular ions on the basis of both charge-to-mass ratio and molecular size
• Because proper preparation of gels is relatively difficult and requires considerable skill, this technique is now rarely used in the clinical laboratory.

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Electrophoresis

• Cellulose Acetate Membrane:-
• Thermoplastic resin made by treating cellulose with acetic anhydride to acetylate the hydroxyl groups.
• Contain about 80% air space within the interlocking cellulose acetate fibers.
• Process takes less than 1 hour & excellent separation without diffusion is achieved.
• Expensive.
• Use:- For separation & identification of Lipoproteins, Isoenzymes & Hemoglobin.

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Electrophoresis

• Agarose:-
• Purified, essentially neutral fraction of agar obtained by separating Agarose from agaropectin.
• Heterogeneous Polysaccharide
• Pore size in Agarose gel is large enough for all proteins to pass.
• So, Separation is based only on the charge-to-mass ratio of the protein.
• Electrophoretic run takes about 90 minutes

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Electrophoresis

• Uses:-
• For separation of Proteins & Nucleic acids
• Used in techniques:-Immunoelectrophoresis & Isoelectric focusing
• Separate RNA & DNA
• Separation of Isoenzymes, Lipoproteins

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Electrophoresis

• Advantages:-
• Show clear fractions after drying
• Serum proteins nicely separated:- five fraction Albumin,α₁, α₂, β, γ-Globulin
• Lipoproteins:- Excellent resolution of:-

β-lipoprotein

pre-β lipoprotein

α-lipoprotein.

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Electrophoresis

• Polyacrylamide Gel:-
• Thermostable, transparent, strong, relatively chemically inert, and depending on concentration can be made in a wide range of pore sizes.
• Separation is based on charge to mass ratio and molecular size (a phenomenon referred to as molecular sieving)
• Serum proteins can be resolved into more fractions than with Agarose gel (> 20 different bands)

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Electrophoresis

• SDS- PAGE:-
• Here proteins are boiled for 1-2 minutes with a denaturing agent, Sodium dodecyl sulphate (SDS)
• The negative charge of SDS will cover the protein molecules, making them strongly negative.
• Then the separation will depend mainly on their molecular size.
• Use:- For molecular weight determination

For assessing the purity of proteins

• Disadvantage:-
• Acrylamide is Potentially Carcinogenic

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�AUTOMATED SYSTEMS �

• Many laboratories are converting to automated systems.
• E.g. Helena SPIFE 3000
• Provides Automated reagent application
• Analysis of 10-100 samples simultaneously

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GENERAL OPERATIONS

• Include Separation, Detection and Quantification
• Separation:-
• Gels are prepared & casted
• 5-7µl of sample is applied using a pipette or comb
• Gel is placed into electrode chamber
• Electrophoresis is performed at specified

current, voltage or power

• Gel is fixed, dried & then stained
• Destained, redried & scanned in densitometer

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Electrophoresis

• Detection:-
• Detected either by staining followed by quantification using a densitometer or by direct measurement using an optical detection system.
• Staining:-
• Proteins- fixed by precipitating them in gel with acetic acid, methanol - prevent diffusion of proteins out of the gel during staining procedure

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Electrophoresis

TYPES OF STAINS USED ALONG WITH THEIR WAVELENGTH OF QUANTIFICATION

• Serum proteins :-
• Amido Black – 640 nm
• Coomasie Brilliant Blue G250 - 595nm
• Coomasie Brilliant Blue R250 - 560nm
• Ponceau S – 520 nm
• Silver stain
• Isoenzymes :-
• Nitrotetrazolium Blue-570 nm

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Electrophoresis

• Lipoprotein zones :-
• Sudan Red 7B - 540 nm
• Oil Red O - 520 nm
• Coomasie brilliant blue R250 - 560nm
• DNA Fragments :-
• Ethidium bromide
• Nucleic acids :-
• Silver stain
• Ethidium bromide
• CSF Proteins :-
• Silver nitrate

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Electrophoresis

• Quantification:-
• Stained zones can be quantified in a Densitometer.
• Measures the absorbance of each fraction as the gel (or other medium) is moved past a photometric optical system and displays an electrophoretogram on a recorder chart or computer display.

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Electrophoresis

TYPES OF ELECTROPHORESIS

• Depending on the nature of supporting

medium:-

• Agarose Gel Electrophoresis
• Polyacrylamide gel electrophoresis
• Cellulose acetate electrophoresis
• Capillary electrophoresis
• Depending on the mode of technique:-
• Disc electrophoresis
• Isoelectric focusing electrophoresis
• Two-dimensional Polyacrylamide gel electrophoresis
• Pulse field electrophoresis
• Immuno electrophoresis
• Capillary electrophoresis
• High voltage electrophoresis

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Electrophoresis

Albumin =>

α₁-globulins =>

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α₂-globulins =>

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β₁-globulins =>

β₂-globulins =>

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γ-globulins =>

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Typical result of an electrophoretic separation of human proteins

Including densitometric evaluation

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Electrophoresis

IMMUNO ELECTROPHORESIS

• Electrophoretic separation is followed by an Antigen- Antibody reaction.
• The precipitation arcs are formed where the antigen and antibody molecules are at 1:1 ratio.
• more than 40 bands

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HIGH VOLTAGE ELECTROPHORESIS

• Usually current is less than 250 volts.
• Since the separation depends on the strength of the current, recent trend is to utilize higher voltages.(400-2000 volts)
• Advantage:- result within ½ an hour
• Use:- for separation of proteins as well as nucleotides from biological fluids.

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CAPILLARY ELECTROPHORESIS

• Gel is taken in a capillary tube of small bore (50-100 microns) and having 100-200 cm in length.
• Nano litre range of sample is injected into the tube. This is connected to buffer and a high voltage power supply of 25,000 volt is applied.
• Within a few minute components are separated.
• Use:- Separation of amino acids, proteins, drugs, vitamins, carbohydrates & nucleotides

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Electrophoresis

ISOELECTRIC FOCUSING

• Based on immobilization of molecules at isoelectric pH.
• In a column, polyacrylamide matrix is filled with ampholytes (substance carrying both positive & negative charge)
• Biological fluids containing proteins or nucleotides are applied. Electricity is then applied.
• The particles migrate, and settle in the matrix where pH matches the isoelectric pH of the particle.

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PULSED FIELD GEL ELECTROPHORESIS(PFGE)

• In conventional gels, the current is applied in a single direction.
• But in PFGE the direction of the current is altered at regular interval. Power is alternately applied to two different pairs of electrodes.
• The electrical field is cycled b/w two direction.
• The frequency of field alterations separate large molecules like DNA with more than 50 kBp size to 400 kBp using appropriate size gels

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TWO DIMENSIONAL ELECTROPHORESIS

• Electrophoresis is done in the first direction based on the charge. Second dimension electrophoresis is based on molecular weight
• Detection of the separated proteins may be done either by autoradiography or Coomassie Blue stain.
• Use:- To study differences in the protein content of cells in genetic disorders. Can also be used to study mutant DNA.

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CLINICAL APPLICATIONS

• Specific protein electrophoresis:-
• Quantitative analysis of specific serum Protein classes such as gamma globulins and albumin.
• Identification & quantitation of hemoglobin and its subclasses.
• Identification of monoclonal proteins such as Bence Jones Gamma globulins in either serum or urine.
• Separation & quantitation of major lipoprotein classes.

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Electrophoresis

• Separation & quantitation of isoenzyme such as creatine kinase, lactate dehydrogenase & alkaline phosphatase into their respective subtypes.
• Used to determine qualitatively the elevation or defiency of specific classes of Immunoglobulins
• Useful in diagnosing various diseases like Multiple Myeloma, Nephrotic syndrome, Sickle cell disease, Cirrhosis of liver etc.

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Electrophoresis

NORMAL SERUM PROTEIN ELECTROPHORESIS

• The pattern of serum protein electrophoresis results depends on the fractions of two major types of proteins : Albumin & Globulin
• Globulins comprises of α₁,α₂,β₁,β₂ and γ globulin fractions
• Thus serum proteins can be resolved into 5-6 bands by electrophoresis
• Albumin has the highest peak & lies closest

towards positive electrode

• The next α₁, α₂, β₁, β₂, γ- globulins lies towards the negative electrode with γ-globulin closest to the negative electrode.

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Electrophoresis

Albumin 50-70 %

α-1 globulin 2-6 %

α-2 gulin 5-11 %

β globlobulin 7-16 %

γ globulin 11-22 %

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Electrophoresis

• Relative concentrations of proteins in serum are :
• Albumin 47 - 71% (3.5 – 5.0 gm/dl)
• Alpha-1 Globulin 2.7- 5.8 % (0.1 – 0.3 gm/dl)
• Alpha-2 Globulin 5.1- 12 % ( 0.6 – 1.2 gm/dl)
• Beta Globulin 8- 12% ( 0.7- 1.3 gm/dl)
• Gamma Globulin 12-23 % (0.6 - 1.8 gm/dl)

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Electrophoresis

• Albumin fraction:-
• Albumin band represents largest protein component
• ↓es in:- Malnutrition, Nephrotic syndrome, Cirrhosis of liver, Burns
• ↑es in:- Dehydration

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Electrophoresis

• Globulin Fraction:-
• α - fraction:- α₁, α₂
• α₁- fraction is composed of α₁-Antitrypsin, TBG (thyroid binding globulin), Transcortin
• ↑es in:-Malignancy & Acute inflammation
• ↓es in:- α₁-Antitrypsin deficiency
• α₂- Fraction is Comprised mainly of α₂-Macroglobulin, Haptoglobin & Ceruloplasmin

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Electrophoresis

• ↑es in:- due to increase in α₂-Macroglobulin seen in Nephrotic Syndrome
• ↓es in:- due to decrease Haptoglobin seen in Hemolytic Anemia (Intravascular)
• β fraction:-
• Has two peaks β₁, β₂- fractions
• β₁ is composed mostly of Transferrin
• β₂ is composed mostly of β-Lipoprotein

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Electrophoresis

• γ- fraction:-
• Composed predominantly of antibodies of IgG type
• ↑es in:- Hodgkin’s Lymphoma, Chronic Lymphatic Lymphoma, Liver diseases & Multiple Myeloma
• Several diseases causes Homogenous spike like peak in the focal region of γ-Globulin called MONOCLONAL GAMMOPATHIES

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Electrophoresis

• Monoclonal gammopathies produce a sharp, spike band & Polyclonal produce diffuse band in region between β & γ regions.
• In Multiple myeloma, a sharp spike is formed and concentration of M band is >3.0 gm/dl
• Thus the quantity of M-protein can help to differentiate between Multiple Myeloma from other monoclonal gammopathies.

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Electrophoresis

• Monoclonal gammopathies are usually associated with a clonal process that is malignant.
• Group of disorders that are characterized by proliferation of a single clone of plasma cells that produce a homogenous M protein
• Polyclonal gammopathies are group of disorders caused by any reactive or inflammatory process & they are usually associated with non-malignant conditions.

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USE OF SERUM PROTEIN

ELECTROPHORESIS

IN DIAGNOSIS OF VARIOUS

DISEASES

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Electrophoresis

• Nephrotic syndrome:-
• There is increase permeability of glomeruli to proteins so that all proteins except very big molecules are lost through urine. So, α₂ fraction containing macroglobulin will be prominent.

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Electrophoresis

• Liver cirrhosis:-
• Albumin synthesis by the liver is decreased with a compensatory excess synthesis of globulins by reticuloendothelial system so albumin band will be thin with a wide β-fraction
• Sometimes β and γ fractions are fused

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• Acute Inflammation:-
• α-1 and α-2 bands are increased during inflammation due to increased hepatic synthesis of acute phase reactant proteins.

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• CHRONIC INFLAMMATION:-
• Immunoglobulin synthesis by the antigen activated T lymphocytes transformed to Plasma cells is demonstrated by the Polyclonal gamma band

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Electrophoresis

• MONOCLONAL GAMMOPATHY:-
• An unusual sharp band in the gamma region suggests the presence of homogenous Immunoglobulin & thus malignant proliferation of plasma cells from a single clone of cell in contrast to the broad gamma band seen in chronic inflammation

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Electrophoresis

Band of fibrinogen in γ globulin region which might be misidentified as ‘M’ band. Therefore plasma is not to be used.

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• α-1 Antitrypsin deficiency:-
• As α -1 antitrypsin is the major component α-1 band there is deficiency of α-1 band

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COMMON USES �

• Separation and quantitation of serum proteins i.e. albumin, α-1 globulin, α-2 globulin, β globulin and γ globulins
• Detection and quantitation of hemoglobin variants for diagnosis of haemoglobinopathies. e.g. sickle cell anemia
• Separation and quantitation of lipoproteins into sub fractions i.e. α-lipoprotein (HDL), pre-β lipoprotein (VLDL) and β lipoprotein (LDL).
• Separation and quantitation of iso-enzymes to detect elevation in a relatively tissue specific iso-enzyme. e.g. lactate dehydrogenase (LDH), alkaline Phosphatase (ALP), creatine kinase (CK) etc.
• Immunoelectrophoresis: For detection of deficiency of specific classes of immunoglobulins.
• Western blot for identification of hepatitis B and HIV virus infections
• Southern blot for identification of specific nucleic acid (DNA, RNA) chain sequence.

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ELECTROPHORESIS

• Definition
• Theory of electrophoresis
• Factors affecting rate of migration
• Instrumentation:- power supply, buffers, support media
• Types of electrophoresis
• Clinical applications:- Nephrotic syndrome, Cirrhosis of liver, Acute & Chronic inflammation, Multiple myeloma, α-1 Antitrypsin deficiency

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INSTRUMENTATION

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THANK YOU

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