This document provides an overview of electrophoresis techniques. It defines electrophoresis as the migration of charged solutes in a liquid medium under an electrical field. It then discusses various electrophoresis techniques including zone electrophoresis, isotachophoresis, agarose gel electrophoresis, cellulose acetate electrophoresis, polyacrylamide gel electrophoresis, isoelectric focusing, and two-dimensional electrophoresis. It provides details on the principles, methods, applications and considerations for each technique.

1. ELECTROPHORESIS
1. Definitions
2. Theory of Electrophoresis
3. Electrophoretic Technique
4. General Procedures
5. Types of Electrophoresis
6. Technical Considerations

2. Gel electrophoresis is a widely used technique for the analysis
of nucleic acids and proteins. Agarose gel electrophoresis is
routinely used for the preparation and analysis of DNA.
Gel electrophoresis is a procedure that separates molecules on
the basis of their rate of movement through a gel under the
influence of an electrical field.
We will be using agarose gel electrophoresis to determine the
presence and size of PCR products.
Agarose Gel Electrophoresis

3. 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
(-)

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

5. Isotachophoresis
• Migration of small ions
• Discontinuous electrolyte system
– leading electrolyte (L- ions, high mobility)
&
– 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

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. 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

9. 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

10. 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

11. 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)

12. 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

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

14. 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

15. 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

16. 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

17. 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

18. 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

19. • 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

20. Denaturing PAGE/SDS-PAGE

21. What is SDS-PAGE?
• Sodium Dodecyl Sulfate Polyacrylamide Gel
Electrophoresis
• A procedure to separate proteins and
determine their Molecular Weights.

22. What is so special about SDS?
• SDS is a negatively charged detergent.
• Disrupts secondary and tertiary protein
structures by breaking hydrogen bonds and
unfolding protein.
• ‘Masks’ charge on protein so that all proteins
act the same as regards charge.
• Prevents protein aggregation.
• Prevents protein shape from influencing gel
run.

25. (i) 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

26. (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

27. Native gradient PAGE example
Zavialov et al. Mol. Microbiol. 2002
Native 4-15% gradient PAGE

28. 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

29. 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)

30. 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)

32. Medium pH range
(pH 4-7)
116
97
81
66
55
45
30
21
14
kDa
pI
4 7
5 6

33. Narrow pH range (1 pH unit)
5.5 6.0
5.0
4.5
4.0
116
66
97
55
81
30
45
21
14
pI
MW
(kDa)
(4.5-5.5)
(4.0-5.0) (5.0-6.0)

34. SDS-PAGE buffers and Solutions
Resolving buffer:
Stacking buffer:
10% APS:
10X SDS-PAGE Tris-Glycine-SDS running buffer:
3X SDS-PAGE loading buffer:
30% 37.5:1 acrylamide/bisacrylamide solution:

35. Southern blot
(DNA)
Northern blot
(RNA)
Western blot
(Protein)
Eastern blot
(???)
Immunoblotting

36. IMMUNOASSAYS
1. Basic Concepts & Definitions
2. Measurement of Antibody Affinity
3. Quantitative Methods – competitive &
noncompetitive assays

37. 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

38. 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

39. 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

40. 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

41. 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

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

43. 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*

44. (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

45. b. Noncompetitive Immunoassays
• Used when have excess reagent
i. 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
iv. Determine bound label ->
[Ag*] or its activity is  [Ag]