electrophoresis overview. electrophoresis definition – refers to the migration of charged solutes...
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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
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