1. ELECTROPHORESIS Definitions Theory of Electrophoresis
Electrophoretic Technique
General Procedures
Types of Electrophoresis
Technical Considerations
NIKAM N.D.
DEPARTMENT OF CHEMISTRY

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 -> 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 thru’out
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 -> each type of ion arrange between L and T ions -> 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 < pI
-> ampholyte/zwitterion has overall +ve charge
Solution with pH > pI
-> ampholyte has overall –ve charge
Under an electric field
-> cations/overall +ve migrate to cathode
-> 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 -> holds buffer
Platinum or carbon electrode -> connected to power supply
Electrophoresis support -> with wicks to contact buffer
Cover -> minimize evaporation (Fig 7-1)

8. 3b. Power Supplies
Power pack: supply current between electrodes
Flow of current -> Heat produced
increase in migration rate -> broadening of separated samples
formation of convection currents -> mixing of separated samples
thermal instability of heat sensitive samples
water loss -> concn of ions -> decrease of buffer viscosity -> decrease in resistance
To minimize problems: use constant-current power supply

9. 3c. Buffers
To carry applied current & to fix the pH
=> determine electrical charge & extent of
ionization => which electrode to migrate
Ionic strength of buffer
thickness of ionic cloud -> migration rate -> sharpness of electrophoretic zones
[ion]  -> ionic cloud  -> 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. -> dry or place in fixative -> treat with dye-fixative
cont. Separation
Separate component using constant voltage or constant current for length of time
Remove support, then
-> dry or place in fixative
-> treat with dye-fixative
-> wash excess dye
-> 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 -> large pore size
higher concns -> small pore size
Serum proteins, Hb variants, lactate dehydrogenase, CK isoenzymes, LP fractions
Pure agarose - does not have ionizable groups -> no endosmosis

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

17. 5b. Cellulose Acetate Electrophoresis (CAE)
Cellulose + acetic anhydride -> CA
Has 80% air space -> 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. load sample about 1/3 way along strip
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 -> 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
-> pour small-pore separation gel
-> large-pore spacer gel cast on top
-> large-pore monomer solution + ~3ul sample
on top of spacer gel
Electrophoresis
-> all protein ions migrate thru large-pore gels
-> concentrate on separation gel
-> separation due to retardation of some
proteins

20. Average pore size in 7.7% PAGE separation gel about 5nm
-> allow most serum proteins to migrate
-> impedes migration of large proteins eg
fibrinogen, 1-lipoprotein, 2-macroglobulin
Advantages:
thermostable, transparent, strong, chemically inert
wide range of pore sizes
uncharged -> 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 -> opens into rod-shaped structures -> separate based on size
Use:
To assess purity of protein
To determine MW of protein

22. (ii) Native PAGE
Use non-denaturing conditions -> no SDS or -mercaptoethanol -> 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 => charge = 0
pI of protein confined in narrow pH range -> sharp protein zones
Method:
use horizontal gels on glass/plastic sheets
introduce ampholytes into gel -> create pH gradient

24. apply a potential difference across gel
cont. IEF Method
apply a potential difference across gel
anode -> area with lowest pH
cathode -> 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 -> in large-pore medium
-> ampholytes to yield pH gradient
2nd D using molecular weight-dependent EP
-> in polyacrylamide -> linear or gradient
O’Farrell 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 -> adsorb hydroxyl ions -> negative charge
Negative charge on support attract positive ions in solution -> Stern potential
As  distance from -ve charge surface
->  no. of –ve ions -> zeta () potential
-> eventually no. +ve ions = -ve ions (Fig 7-8)

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

28. In cellulose acetate & agarose gel Reduce endosmosis by
cont. Endosmosis
In cellulose acetate & agarose gel
Reduce endosmosis by
removing/modifying charged groups on support
adding of sucrose or sorbitol -> 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 -> 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 -> bind tightly to Ag -> 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 -> association & dissociation
Ag + Ab <-> AgAb
Law of mass action:
Rate of rxn  to concn of reactants
ka[Ag][Ab] = kd[AgAb]
K = ka/kd = [AgAb]/ [Ag][Ab]
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 => 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. Labeled Immunochemical Assays
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* <-> Ab:Ag + A-Ag*

37. (ii) Sequential Competitive Assay
Step 1: unlabeled Ag mixed with excess Ab
-> binding allowed to reach equilibrium
Step 2: Ag* added sequentially -> equilibrate
After separation -> det bound Ag* -> calculate [Ag]
Larger fraction of Ag bound to Ab than in simultaneous assay
If k1 >> k2 ->  in Ab:Ag ->  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 -> wash other proteins
iii. Add labeled Ab (conjugate) -> reacts with
bound Ag
Determine bound label ->
[Ag*] or its activity is  [Ag]