Electrophoresis

Overview

Electrophoresis

• Definition – refers to the migration of charged solutes or

particles in a liquid medium under the influence of an electrical field

– Iontophoresis • limited to the migration of small ions

• free solution or moving boundary method

a typical electrophoresis apparatus

• Zone Electrophoresis– Migration of charged molecules– usually in a porous supporting medium• cellulose acetate sheets or agarose gel film

– generates an electrophoretogram

• Support medium• Protein zones are visualized by – staining with a protein-specific stain

• Support medium can be dry and record • Quantitation – Densitometer

• Solutes of interest – Proteins in serum, urine, cerebrospinal fluid

• Background electrolyte (buffer)

isotachophoresis

• applies specifically to the migration of small ions• no background electrolyte (buffer) is mixed with

the sample• leading electrolyte solution – contains ions that are faster than any in the sample

• Trailing solution – slower than any in the sample

• Sample between leading and Trailing electrolyte

isotachophoresis

• Applications– separation of small anions and cations, organic

and amino acids, peptides, nucleotides, nucleosides, and proteins.

• The rate of migration is dependent on – Net electrical charge – Size and shape of the molecule– Electric field strength– Properties of the supporting medium– Temperature of operation

• resisting force (counter-force) – Ionic radius of the solute – the viscosity of the buffer solution

• electrophoretic mobility– the rate of migration (cm/s) per unit field strength – expressed by the symbol µ – directly proportional to the net charge – inversely proportional to the size of the molecule – inversely proportional to the viscosity of the

electrophoresis medium

• Factors that can affect mobility– Temperature– Ionic strength – Rate of endosmotic flow– Average pore size of support medium– Point of sample application– wick flow • Drying effect

– flow of buffer from both directions

Instrumentation and Reagents

• Power Supplies – operation at either constant current– constant voltage– constant power

• flow of current through a medium – Resistance, production of heat– Heat• thermal agitation of all dissolved ions

– increase in both the migration rate– and the rate of evaporation of water

• The water loss– increase in ion concentration• decrease in resistance

– increases the conductance of the system

• To minimize these effects on the migration rate– it is best to use a constant-current power supply

Pulsed-power or pulsed-field

• periodically change the orientation of the applied field

• molecules must reorient • new field direction to fit through the pores in

gel• reorientation time depends on molecular size• net migration becomes a function of the

frequency of field alteration

• separation of very large molecules– Such as DNA fragments greater than 50 kilobases

Buffers

• they carry the applied current• fix the pH at which electrophoresis is carried out• determine the kind of electrical charge on the solute• the extent of ionization of the solute • they determine the electrode toward which the

solute will migrate • The buffer’s ionic strength– concentration of ions

• With increasing– molecule becomes more hindered in its movement

Buffers• sharpness of the electrophoretic zones• High ionic strength buffer

– reduction in resistance – leads to increased current

• excessive heat – leads to denaturation of heat-labile proteins – degradation of other components

– sharper band separations

• Ionic strength – ion concentration– the charge on the ion

• most widely used buffers– barbital buffers – Tris-boric acid-EDTA buffers

Protein Stains

• visualize and locate the separated protein fractions

• Dyes commonly used in electrophoresis table– Amido Black (Naphthol Blue Black)– Ponceau S– reactivity toward carrier ampholytes

• not suitable for polyacrylamide gel-isoelectric focusing

• The amount of dye taken up by the sample– type of protein– degree of denaturation of the proteins

Protein Stains

Support Media

• solutions – such as a sucrose gradient

• insoluble gels– sheets, slabs, or columns of starch, agarose, or

polyacrylamide,membranes of cellulose acetate

Automated Systems

• prepackaged gels, sample application through electrophoresis, staining, scanning of gels, and computation of results.

• partially automated– ability to process multiple gels of different

compositions – simultaneous processing of seven samples by

using multiple capillaries

General Procedures• a hydrated support material– freshly prepared agarose gel– wetted cellulose acetate

• Excess buffer removed from the support surface by blotting

• bubbles not be present • support is placed in contact with buffer • Sample is applied to the support • and electrophoresis is conducted using either

constant voltage or constant current

General Procedures

• rapidly dried or placed in a fixative • treated with a dye-fixative reagent (staining)• washing out excess dye• the support is dried – agarose

• placed in a clearing agent • cellulose acetate membranes

Detection and Quantitation

• Densitornetry– moved past a measuring optical system

• the area under each peak

• report the results– percentage of each fraction present– in terms of absolute concentration

• Reliable quantitation of stained zones– requires light of an appropriate wavelength– a linear response from the instrument– a transparent background

Detection and Quantitation

• agarose gels– satisfies the requirement for a clear background

• problems associated with densitometry– differences in quantity of stain taken up by

individual proteins – differences in protein zone sizes

TYPES OF ELECTROPHORESIS

• Starch Gel Electrophoresis– Separates macromolecular ions on the basis• surface charge • molecular size

– may be used in a horizontal or vertical direction– proper preparation of gels is relatively difficult– rarely used in the clinical laboratory

TYPES OF ELECTROPHORESIS

• Agarose Gel Electrophoresis (AGE)– purified, essentially neutral fraction of agar– a convenient method – applied to the analysis of serum proteins,

hemoglobin variants, lactate dehydrogenase and creatine kinase isoenzymes, lipoprotein fractions

– free of ionizable groups• exhibits little endosmosis • exhibits little background staining

– native clarity

TYPES OF ELECTROPHORESIS

• the agarose surface remain undisturbed– This avoids the surface artifact

• Requires sample volume of 0.6 to 3 µL • Electrophoresis time of 30 to 90 min

TYPES OF ELECTROPHORESIS

• Cellulose Acetate (CAE) – come as dry, opaque, brittle films– the film is soaked in buffer– Characteristics vary with • extent of acetylation• prewashing procedure used by the manufacturer• the additives used• the pore size • thickness of the membrane

TYPES OF ELECTROPHORESIS

• Serum samples (0.3-2.0 µL) are generally applied

• may be made transparent – soaking in a solvent mixture • 95 parts methanol and 5 parts glacial acetic acid

• Advantage– speed of separation 20 min- 1 h– ability to store for long periods

• disadvantage– need for presoaking before use – need clearing the strips prior to densitometry

• largely been replaced by agarose gel

TYPES OF ELECTROPHORESIS

• Disc Electrophoresis– discontinuities in the electrophoretic matrix – layers of gel that differ in composition and pore size– several proteins with the same electrophoretic

mobility• to overcome these deficiencies

– Diffusion,broading

– Polyacrylamide and Starch gel • pore size is controlled by the percent composition • much smaller than that found in agarose gel

TYPES OF ELECTROPHORESIS

• Proteins are separated– on the basis of charge and molecular size • molecular sieving

– may yield 20 or more fractions – to study individual proteins in serum• genetic variants and isoenzymes

TYPES OF ELECTROPHORESIS• Polyacrylamide Gel Electrophoresis (PAGE)

– three different layers of gel• small-pore separation gel • large-pore gel (spacer gel )• a large-pore monomer solution contaming a small amount of serum

– Separation then takes place in the bottom separation gel • Advantage

– Thermostable– Transparent – Strong– relatively chemically inert – can be made in a wide range of pore sizes

• potential carcinogenicity• larger pores; less resistance to the passage of

large molecules• ideally suited to the separation of DNA

fragments up to 20 kilobases • In homogeneous (non-pulsed) electric field

Isoelectric Focusing• The protein migration in a medium possessing a

stable pH gradient • moves to a zone in the medium where the pH is

equal to the isoelectric point (pI) of the protein.• the charge becomes zero • migration ceases• the protein zones are very sharp– diffusion is also counteracted

• acquisition of charge • only 0.02 pH unit

• carrier ampholytes – create buffered zones– high concentrations • a high-voltage power source

– matrix must be cooled

• matrix – polyacrylamide gel• optically clear and supple • large enough pore size

– IgM impeded

• the anode is surrounded by a dilute acid solution

• the cathode by a dilute alkaline solution• Matrix characteristics – Agarose, cellulose acetate • Electroendosmosis-free materials• operating conditions are simple • large pore sizes

Two-Dimensional (2D) Electrophoresis

• charge-dependent IEF electrophoresis in the first dimension

• molecular weight-dependent electrophoresis in the second dimension

• first dimensional in a large-pore medium – Ampholytes are added to yield a pH gradient

• The second dimension is often polyacrylamide in a line ar or gradient format

• Additives – SDS is used in the second dimension– β-mercaptoethanol in the first – SDS in both dimensions and in sample preparation

• Electrophoresis under – Native condition– Denaturing conditions

• Detection method– Autoradiography – Using Coomassie dyes– Radiography– fluorographic analysis

• method limitation– From 7000 polypeptide spots – Autoradiography

• 1100 spots are detected – Using Coomassie dyes

• about 400 polypeptides are detected

• Coomassie dyes – three times more sensitive than Amido Black

• silver staining– 100 times more sensitive than Coomassie dye• Radiography , fluorographic analysis – greatest analytical sensitivity

High-Resolution Electrophoresis

• high-ionic-strength buffer– µ = 0.075

• pH 8.6• admixture of calcium lactate • Support – agarose gel

• temperature control is necessary • Serum proteins resolved into as many as 13 zones

High-Resolution Electrophoresis

• Discontinuous buffer system– different buffer is used in the electrode chambers

from that in the gel• two different pH

– alter relative mobility

Capillary Electrophoresis • electrophoresis are carried out in capillary tube – 10-100 µm diameter – 20 to 200 cm in length – detector at its terminal end – high-voltage power up to 30 kV– mostly made of fused silica (i.e., pure glass),

polyethylene

• Advantage – Improved heat dissipation– Sample volumes

• in the picoliter to nanoliter range

Capillary Electrophoresis • Reduced separation time • Can be fully automated• applications – low- molecular-weight ions to proteins and other

macromolecules– Even uncharged molecules

• minimizing band spreading – Improved resolution

• Due to narrow bore • a variety of detector types can be used

Capillary Electrophoresis

• Detectors– Optical methods • Ultraviolet- visible photometers• Fluorescence

– laser-induced fluorescence

• Refractive index• Chemiluminescence

– Mass spectrometers– Electrochemical detection methods

• Efficient and high-resolution separations– High electric fields

• Produce high Joule heat – non uniform temperature gradients – local changes in viscosity

» subsequent zone broadening

• Control of heat production – reducing the diameter of the capillary tube– reducing the ionic strength of the running electrolyte– reducing the applied voltage

• Charged solutes migrate through a polymer network

• Larger solutes hindered more than small ones• Molecular sieve• Separation using either– Free solution electrophoresis • Macromolecules, such as DNA and SDS-saturated

proteins, cannot be separated without a gel

– or a precast gel

Blotting Techniques

• Southern Blotting – DNA or fragments of DNA are first separated by AGE– fragments are transferred or ‘‘blotted” onto nitrocellulose

or a nylon membrane – detected and identified by hybridization with a labeled,

complementary nucleic acid probe– determining the presence, position, and number of copies

of a gene in a genome

Blotting Techniques

• Northern Blotting– to separate and detect RNAs and RNA fragments – first separated by AGE (agarose gel electrophoresis )

– blotted to an overlying strip of nitrocellulose – detected and identified by hybridization to a

labeled RNA probe

Blotting Techniques

• Western Blotting– to separate, detect, and identify one or more proteins– first separating the individual proteins by SDS PAGE – transferred or “blotted” onto an overlying strip of

nitrocellulose – reacted with a reagent that contains an antibody raised

against the protein of interest

TECHNICAL CONSIDERATIONS

• Electroendosmosis or Endosmosis effect – Support medium

• in contact with water takes on a negative charge– fixed ions

• Formation an ionic cloud – associated cloud of ions

» free to move • the ions in solution are highly hydrated

– current is applied • Movement to the electrode of opposite polarity• movement of solvent and its solutes relative to the fixed support

– preferential movement of water in one direction

TECHNICAL CONSIDERATIONS

• media in which endosmosis is strong– conventional cellulose acetate – conventional agarose gel• Sulfate or carboxylic acid groups

• surface charges are minimal – starch gel – polyacrylamide gel

TECHNICAL CONSIDERATIONS

• Buffers– Good culture media – should be refrigerated – discarded after each run • pH changes resulting from the electrolysis

– For four electrophoretic runs• Switched the polarity after each run• Mix both buffer boxes

TECHNICAL CONSIDERATIONS

• Stain Solution – 100 mL for a combined total of 387 cm2

– Considered faulty • if leaching of stained protein zones occurs in the 5%

acetic acid • Whenever protein zones appear too lightly stained

– stored tightly covered to avoid evaporation

TECHNICAL CONSIDERATIONS

• Sampling– Typical amounts • to cellulose acetate are 0.3 to 1 .6 µL• In PAGE 3 µL (about 210 pg of total protein)• Agarose

– 0.6 to 2.0 µL • depending on the test requirements

– For isoenzyme analysis » as much as 25 µL of a normal serum

– Discontinuities in Sample Application • due to dirty applicators

TECHNICAL CONSIDERATIONS

• Unequal Migration Rates – dirty electrodes• uneven application of the electric field

– Uneven wetting of the gel

TECHNICAL CONSIDERATIONS

• Distorted, Unusual, or Atypical Bands – Zone Distortion • bent applicators• incorporation of an air bubble • Over-application of sample

– overfill the sample well

• excessive drying of the electrophoretic support• excessively wet

– cellulose acetate films or agarose gels

• Unusual Bands – Artifacts – Hemolyzed samples

• an increased β-globulin • unusual band between the α2- and β2-globulins

– A band at the starting point • may be fibrinogen

– The sample should be verified as being serum

– Split zone • α1-, α2-, and β-globulins• split albumin zone

– Widened zone • Albumin, in certain medications

Atypical Bands• the result of binding – In an isoenzyme pattern

• by an immunoglobulin • Irregular but sharp protein zone at the starting point– artifact

• lacks the regular, somewhat diffuse appearance – Fibrinogen– Denatured protein

• deteriorated serum – Damage done to the cellulose acetate

• Paraprotein

Evaluation of electrophoretic quality

• Include a control serum