ELECTROPHORESIS

1

• ELECTROPHORESIS
• Definitions
• Theory of Electrophoresis
• Electrophoretic Technique
• General Procedures
• Types of Electrophoresis
• Technical Considerations
• Ref Burtis Ashwood Tietz Fundamentals/Textbook
  

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• 1. DEFINITIONS
• Electrophoresis
• Migration of charged solutes in a liquid medium
  under an electrical field
• Many biological molecules have ionisable groups
  eg. amino acids, proteins, nucleotides, nucleic
  acids
• Under an electric field -gt charged particles
  migrate to anode () or cathode (-)

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• Zone Electrophoresis
• Migration of charged molecules
• Support medium
• porous eg. CA or agarose
• can be dried kept
• Same pH field strength thruout
• Separation based on electrophoretic mobility
• Separates macromolecular colloids eg. proteins in
  serum, urine, CSF, erythrocytes nucleic acids

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• Isotachophoresis
• Migration of small ions
• Discontinuous electrolyte system
• leading electrolyte (L- ions)
• trailing electrolyte (T- ions)
• Apply sample solution at interphase of L T
• Apply electric field -gt each type of ion arrange
  between L and T ions -gt discrete zones
• Separates small anions, cations, organic amino
  acids, peptides, nucleotides, nucleosides,
  proteins

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• 2. THEORY of ELECTROPHORESIS
• Many biological molecules exist as
• (a) cations or (b) anions
• Solution with pH lt pI
• -gt ampholyte/zwitterion has overall ve charge
• Solution with pH gt pI
• -gt ampholyte has overall ve charge
• Under an electric field
• -gt cations/overall ve migrate to cathode
• -gt anions/overall -ve migrate to anode

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• Rate of migration depends on
• Net electrical charge of molecule
• Size shape of molecule
• Electric field strength
• Properties of supporting medium
• Temperature of operation

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• 3. ELECTROPHORETIC TECHNIQUE
• 3a. Instrumentation Reagents
• Buffer boxes with buffer plates -gt holds buffer
• Platinum or carbon electrode -gt connected to
  power supply
• Electrophoresis support -gt with wicks to contact
  buffer
• Cover -gt minimize evaporation (Fig 7-1)

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• 3b. Power Supplies
• Power pack supply current between electrodes
• Flow of current -gt Heat produced
• increase in migration rate -gt broadening of
  separated samples
• formation of convection currents -gt mixing of
  separated samples
• thermal instability of heat sensitive samples
• water loss -gt concn of ions -gt decrease of buffer
  viscosity -gt decrease in resistance
• To minimize problems use constant-current power
  supply

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• 3c. Buffers
• To carry applied current to fix the pH
• gt determine electrical charge extent of
• ionization gt which electrode to
  migrate
• Ionic strength of buffer
• thickness of ionic cloud -gt migration rate -gt
  sharpness of electrophoretic zones
• ion ? -gt ionic cloud ? -gt movement of molecules
  ?
• Barbital buffers Tris-boric acid-EDTA buffers

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• 3d. Protein Stains
• To visualize/locate separated protein fractions
• Dyes amount taken up depends on
• Type of protein
• Degree of denaturation of proteins by fixing
  agents
• Types of stains Table 7-1

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• 4. GENERAL PROCEDURES
• 4a. Separation
• Place support material in EP chamber
• Blot excess buffer from support material
• Place support in contact with buffer in electrode
  chamber
• Apply sample to support

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• cont. Separation
• Separate component using constant voltage or
  constant current for length of time
• Remove support, then
• -gt dry or place in fixative
• -gt treat with dye-fixative
• -gt wash excess dye
• -gt dry (agarose) or put in clearing agent (CA
• membs)

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• 4b. Detection Quantitation
• Express as
• of each fraction present or
• absolute concn
• By densitometry
• electrophoretic strip moved past an optical
  system
• absorbance of each fraction displayed on recorder
  chart

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• 5. TYPES OF ELECTROPHORESIS
• Agarose Gel Electrophoresis
• b. Cellulose Acetate Electrophoresis
• c. Polyacrylamide Gel Electrophoresis
• d. Isoelectric Focusing
• e. Two-dimensional Electrophoresis

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• 5a. Agarose Gel Electrophoresis (AGE)
• Use agarose as medium
• low concns -gt large pore size
• higher concns -gt small pore size
• Serum proteins, Hb variants, lactate
  dehydrogenase, CK isoenzymes, LP fractions
• Pure agarose - does not have ionizable groups -gt
  no endosmosis

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• Cont. AGE
• Advantages
• low affinity for proteins
• shows clear fractions after drying
• low melting temp -gt reliquify at 65oC
• Disadvantage
• poor elasticity
• -gt not for gel rod system
• -gt horizontal slab gels

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• 5b. Cellulose Acetate Electrophoresis (CAE)
• Cellulose acetic anhydride -gt CA
• Has 80 air space -gt fill with liquid when soaked
  in buffer
• Can be made transparent for densitometry
• Advantages
• speed of separation
• able to store transparent membranes
• Disadvantages
• presoaking before use
• clearing for densitometry

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• cont. CAE
• Method
• wet CA in EP buffer
• load sample about 1/3 way along strip
• stretch CA in strips across a bridge
• place bridge in EP chamber -gt strips dip directly
  into buffer
• after EP, stain, destain, visualise proteins
• For diagnosis of diseases
• change in serum protein profile

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• 5c. Polyacrylamide Gel Electrophoresis (PAGE)
• Tubular-shaped EP cell
• -gt pour small-pore separation gel
• -gt large-pore spacer gel cast on top
• -gt large-pore monomer solution 3ul sample
• on top of spacer gel
• Electrophoresis
• -gt all protein ions migrate thru large-pore gels
• -gt concentrate on separation gel
• -gt separation due to retardation of some
• proteins

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• Average pore size in 7.7 PAGE separation gel
  about 5nm
• -gt allow most serum proteins to migrate
• -gt impedes migration of large proteins eg
• fibrinogen, ?1-lipoprotein,
  ?2-macroglobulin
• Advantages
• thermostable, transparent, strong, chemically
  inert
• wide range of pore sizes
• uncharged -gt no endosmosis
• Disadvantages
• carcinogenic

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• Denaturing PAGE/SDS-PAGE
• Boil sample for 5 mins in sample buffer
  containing ?-mercaptoethanol SDS
• ?-mercaptoethanol reduce disulfide bridges
• SDS binds strongly to denatures proteins
• Proteins denatured -gt opens into rod-shaped
  structures -gt separate based on size
• Use
• To assess purity of protein
• To determine MW of protein

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• (ii) Native PAGE
• Use non-denaturing conditions -gt no SDS or
  ?-mercaptoethanol -gt proteins not denatured
• Proteins separate based on
• different electrophoretic mobilities
• sieving effects of gel
• Use
• to obtain native protein/enzyme
• to study biological activity

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• 5d. Isoelectric Focusing
• To separate amphoteric cpds eg. proteins
• Proteins moves to zone where
• pH medium pI protein gt charge 0
• pI of protein confined in narrow pH range -gt
  sharp protein zones
• Method
• use horizontal gels on glass/plastic sheets
• introduce ampholytes into gel -gt create pH
  gradient

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• cont. IEF Method
• apply a potential difference across gel
• anode -gt area with lowest pH
• cathode -gt area with highest pH
• proteins migrate until it arrives at pH pI
• wash with fixing solution to remove ampholytes
• stain, destain, visualise
• Separations of proteins with 0.01 to 0.02pH unit
  differences (Fig 7-4)

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• 5e. Two-Dimensional (2D) EP (ISO-DALT)
• 1st D using IEF EP -gt in large-pore medium
• -gt ampholytes to yield pH gradient
• 2nd D using molecular weight-dependent EP
• -gt in polyacrylamide -gt linear or gradient
• OFarrell method
• use ?-mercaptoethanol (1st D) SDS (2nd D)
• Detect proteins using Coomassie dyes, silver
  stain, radiography, fluorography
• Separates 1100 spots (autoradiography)

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• 6. TECHNICAL CONSIDERATIONS
• Electroendosmosis/Endosmosis
• Support in contact with water -gt adsorb hydroxyl
  ions -gt negative charge
• Negative charge on support attract positive ions
  in solution -gt Stern potential
• As ? distance from -ve charge surface
• -gt ? no. of ve ions -gt zeta (?) potential
• -gt eventually no. ve ions -ve ions (Fig 7-8)

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• When apply current to system
• -gt -ve charges on support remain fixed
• -gt cloud of ions in solution move to electrodes
• -gt ions highly hydrated gt as ionic cloud moves,
  
• 
  solvent also moves
• Movement of solvent relative to fixed support gt
  endosmosis
• Movement of water in one direction
• Macromolecules moving in opposite direction
  oppose flow of hydrated ve ions -gt may remain
  immobile or be swept to opposite pole

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• cont. Endosmosis
• In cellulose acetate agarose gel
• Reduce endosmosis by
• removing/modifying charged groups on support
• adding of sucrose or sorbitol -gt increase
  osmolality

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• IMMUNOASSAYS
• Basic Concepts Definitions
• Measurement of Antibody Affinity
• Quantitative Methods competitive
  noncompetitive assays
• Ref Burtis Ashwood Tietz Fundamentals/Textbook
  
• Jan Klein Vaclav Horejsi Immunology
  (1997)
• Coleman Lombard Sicard Fundamental Imm
  (1992)
• Gary D. Christian Analytical Chem (1994)

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• 1. BASIC CONCEPTS DEFINITIONS
• Immunoassay use of antibodies to detect analyte
• 1a. Antibodies
• Immunoglobulins that bind to Antigens
• 5 classes IgG, IgA, IgM, IgD, IgE
• 1b. Immunogen
• Protein or a substance coupled to a carrier
• When introduced into foreign host -gt induce Ab to
  form
• 1c. Antigen
• Any material which can react with Ab
• May not induce Ab formation

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• 1d. Antigen-Antibody Binding
• Ab molecules have specific binding sites -gt bind
  tightly to Ag -gt cause pptn/neutralization/ death
• Binding of Ag to Ab due to
• van der Waals forces
• hydrophobic interactions
• charged group attractions
• Can measure Antibody affinity strength of
  binding between Ab Ag

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• 2. MEASUREMENT OF ANTIBODY AFFINITY
• Binding of Ag to Ab is reversible -gt association
  dissociation
• Ag Ab lt-gt AgAb
• Law of mass action
• Rate of rxn ? to concn of reactants
• kaAgAb kdAgAb
• K ka/kd AgAb/ AgAb
• where K is equilibrium constant or affinity
  constant

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• r/c nK rK
• r no. of molecules of bound Ag per Ab molecule
• c concn of free Ag
• n valency of Ab
• Plot r/c vs r gt Scatchard Plot
• Straight line with slope k
• x intercept gives n
• y intercept gives nK
• K (liters/mole) measures affinity of complex

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• Why measure Affinity of an Antibody?
• To assess Ab specificity
• It influences the functional efficiencies of Abs
• eg. high-affinity Abs are very dependable for
• various applications
• Diagnostic
• Therapeutic
• Analytical

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• 3. QUANTITATIVE METHODS
• Read Understand from Tietz Fundamentals
• Radial Immunidiffusion Immunoassay
• Electroimmunoassay
• Turbidimetric Nephelometric Assays
• Labeled Immunochemical Assays

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• COMPETITIVE vs NONCOMPETITIVE RXNS
• A. Competitive Immunoassays
• Used when have limited reagents (Ag)
• (i) Simultaneous Competitive Assay
• Labels Ag (Ag) unlabeled Ag compete for
  binding to Ab
• The probability of Ab binding to Ag is inversely
  ? to Ag
• Ab Ag Ag lt-gt AbAg A-Ag

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• (ii) Sequential Competitive Assay
• Step 1 unlabeled Ag mixed with excess Ab
• -gt binding allowed to reach
  equilibrium
• Step 2 Ag added sequentially -gt equilibrate
• After separation -gt det bound Ag -gt calculate
  Ag
• Larger fraction of Ag bound to Ab than in
  simultaneous assay
• If k1 gtgt k2 -gt ? in AbAg -gt ? in Ag binding
• Provide two- to four- fold improvement in
  detection limit

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• b. Noncompetitive Immunoassays
• Used when have excess reagent
• Immobilization of Ab to support
• Passively adsorption or bind covalently
• Direct or indirect attachment (Table 9-3)
• ii. Ag allowed to react with Ab -gt wash other
  proteins
• iii. Add labeled Ab (conjugate) -gt reacts with
• 
  bound Ag
• Determine bound label -gt
• Ag or its activity is ? Ag