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ELECTROPHORESIS

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ELECTROPHORESIS Definitions Theory of Electrophoresis Electrophoretic Technique General Procedures Types of Electrophoresis Technical Considerations – PowerPoint PPT presentation

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Title: ELECTROPHORESIS


1
  • ELECTROPHORESIS
  • Definitions
  • Theory of Electrophoresis
  • Electrophoretic Technique
  • General Procedures
  • Types of Electrophoresis
  • Technical Considerations
  • Ref Burtis Ashwood Tietz Fundamentals/Textbook

2
  • 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 (-)

3
  • 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

4
  • 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

5
  • 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

6
  • Rate of migration depends on
  • Net electrical charge of molecule
  • Size shape of molecule
  • Electric field strength
  • Properties of supporting medium
  • Temperature of operation

7
  • 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)

8
  • 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

9
  • 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

10
  • 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

11
  • 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

12
  • 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)

13
  • 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

14
  • 5. TYPES OF ELECTROPHORESIS
  • Agarose Gel Electrophoresis
  • b. Cellulose Acetate Electrophoresis
  • c. Polyacrylamide Gel Electrophoresis
  • d. Isoelectric Focusing
  • e. Two-dimensional Electrophoresis

15
  • 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

16
  • 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

17
  • 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

18
  • 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

19
  • 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

20
  • 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

21
  • 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

22
  • (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

23
  • 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

24
  • 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)

25
  • 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)

26
  • 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)

27
  • 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

28
  • cont. Endosmosis
  • In cellulose acetate agarose gel
  • Reduce endosmosis by
  • removing/modifying charged groups on support
  • adding of sucrose or sorbitol -gt increase
    osmolality

29
  • 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)

30
  • 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

31
  • 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

32
  • 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

33
  • 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

34
  • 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

35
  • 3. QUANTITATIVE METHODS
  • Read Understand from Tietz Fundamentals
  • Radial Immunidiffusion Immunoassay
  • Electroimmunoassay
  • Turbidimetric Nephelometric Assays
  • Labeled Immunochemical Assays

36
  • 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

37
  • (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

38
  • 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
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