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ELECTROPHORESIS
1.
2.
3.
4.
5.
6.
Definitions
Theory of Electrophoresis
Electrophoretic Technique
General Procedures
Types of Electrophoresis
Technical Considerations
Agarose Gel Electrophoresis
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.
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
(-)
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
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
• Rate of migration depends on:
– Net electrical charge of molecule
– Size & shape of molecule
– Electric field strength
– Properties of supporting medium
– Temperature of operation
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)
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
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
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
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)
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
5. TYPES OF ELECTROPHORESIS
a. Agarose Gel Electrophoresis
b. Cellulose Acetate Electrophoresis
c. Polyacrylamide Gel Electrophoresis
d. Isoelectric Focusing
e. Two-dimensional Electrophoresis
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
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
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
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
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
• 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, 2macroglobulin
• Advantages:
– thermostable, transparent, strong,
chemically inert
– wide range of pore sizes
– uncharged -> no endosmosis
• Disadvantages:
– carcinogenic
Denaturing PAGE/SDS-PAGE
What is SDS-PAGE?
• Sodium Dodecyl Sulfate Polyacrylamide Gel
Electrophoresis
• A procedure to separate proteins and
determine their Molecular Weights.
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.
(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 rodshaped structures -> separate based on
size

Use:
– To assess purity of protein
– To determine MW of protein
(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
Native gradient PAGE example
Native 4-15% gradient PAGE
Zavialov et al. Mol. Microbiol. 2002
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
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)
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)
Medium pH range
(pH 4-7)
pI
4
kDa
116
97
81
66
55
45
30
21
14
5
6
7
Narrow pH range (1 pH unit)
(4.5-5.5)
(4.0-5.0)
(5.0-6.0)
pI
4.0
116
97
81
66
55
45
MW
(kDa)
30
21
14
4.5
5.0
5.5
6.0
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:
Immunoblotting
Northern blot
(RNA)
Western blot
(Protein)
Eastern blot
(???)
Southern blot
(DNA)
IMMUNOASSAYS
1. Basic Concepts & Definitions
2. Measurement of Antibody Affinity
3. Quantitative Methods – competitive &
noncompetitive assays
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
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
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
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
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
3. QUANTITATIVE METHODS
• Read & Understand from Tietz Fundamentals:
– Radial Immunidiffusion Immunoassay
– Electroimmunoassay
– Turbidimetric & Nephelometric Assays
• Labeled Immunochemical Assays
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*
(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
b. Noncompetitive Immunoassays
•
i.
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
iv. Determine bound label ->
[Ag*] or its activity is  [Ag]