Download pH of the electrophoresis buffer

Electrophoresis
• Separation according to migration of charged
particles in electric field
• Electrolysis: Chemical decomposition
supplemented by current (components reach
electrode)
• F=vf=qE
V=Velocity, E=Electric field, q=Charge
f=Frictional coefficient
Electrophoresis
Electrophoretic mobility
• Electrophoretic mobility μ = v / E = q / f
– V=Velocity, E=Electric field, q=Charge
f=Frictional coefficient
• Depends on
– Particle property :Surface, charge, density &
size
– Solution properties: Ionic strength, pH,
Conductivity,viscosity
– Temperature & Voltage
Types of electrophoresis- Moving
boundary Electrophoresis
Moving boundary
Electrophoresis
• U shaped
• Multiple moving boundaries due to different
components
• Local changes in refractive index measured
Zonal Electrophoresis
Zonal electrophoresis
• Complete separation achieved
• Discrete zones formed
• 3 ways to stabilize components
–
–
–
–
(i)Use of support material:
Filter paper
Membrane
Gel
Zonal electrophoresis
– (ii)Density gradient stabilization:
- Sucrose,glycerol,ethylene glycerol
- Prevents convection current
(iii)Free zone electrophoresis
• Rotation of vessel during run
• Continuous film of buffer
perpendicular to electric field
Isoelectric Focusing/Steady state
electrophoresis
Steady State electrophoresis/
Isoelectric focusing
•
•
•
•
Separates molecules by charge
Performed in gel
Distributed over medium that has pH gradient
Molecules move towards opposite polarity under
current
• Settle at Isoelectric point :Molecule no longer has
a net electric charge (due to the protonation or
deprotonation of the associated functional groups)
Support media:
1. Filter paper: paper electrophoresis (Principle same
as paper electrophoresis)
Moist paper is placed on a supporting rack, samples
are applied with a capillary applicator, paper is
loaded onto the apparatus with its ends dipping into
an electrode vessel containing buffer, and covered
to prevent
drying
at the
end of run,
paper is
carefully taken out and
dried, and examined by
appropriate detection
technique
Disadvantage
• Thin paper: Tear
• Thick paper: Boundaries between the zones distort
• OH group of cellulose interact with biomolecules
and results in ‘tailing’
• water absorbing and electrical conductivity capacity
of paper depends on its content (96% -cellulose)
• Electro osmosis
Electro osmosis
•
dissociation of COOH group(cellulose matrix)
generation of (H3O)+ ionMoves (electric
field)
•
A correction factor for electrophoretic mobility:
•
distance between the sample origin and the
position of the blue dextran (does not ionize)
•
20V cm-1 – zone diffusion of small molecules
•
200 V cm-1 – High voltage EP
2. Cellulose Acetate Membrane: Advantages over paper
•
OH groups of cellulose are esterified by acetylation
• spectrophotometric determination:Use of mineral oil
of RI equals to that of the membrane;
• Easy dissolution: Recovery of separated sample
• Procedure similar to paper
3. Gel Media:
Network of polymer molecules
surrounded and penetrated by
buffer
space between the gel molecules
are the porous
frictional resistance to movement
(1) relative size of gel pore
(2)radius of the molecule;
exerts a sieving effect
a) Starch Gels:
• earlier work with proteins used starch as support
• cheap, easy and reasonably quick
Disadvantage
• pore size can vary within only a narrow range
(determined by the starch conc)
• contains some negatively charged side-chain:
hinder separation
b) Polyacrylamide Gels:
Made by cross linking acrylamide with N, N’methylenebisacrylamide
Requires an initiator (APS of PPS) for chemical
polymerization & light and ribofalvin for
photopolymerization
Crosslinking reagent is N, N, N’, N’tetramethylethylenediamine (TEMED)
Ratio of cross-linking agent to acrylamide is a
factor determines the pore size of the gel
Can be tube gel or slab gel
1. Tube Gel
2. Slab Gel EP: Advantage; heat dissipation is better,
higher resolving power; can be used horizontally
(when gel conc is low) or vertically
Variation of PAGE:
a) Disc Electrophoresis:
consist of two different containers layered one on
top of the other
Stacking gel:large pore,
no molecular sieve
effect
acrylamide conc 2.5%,
pH and ionic strength
lower than running gel
b) SDS-PAGE:
PAGE:Separation depends on the net charge,size and
shape
SDS-PAGE: Difference in net charges eliminated
by complexing with anionic detergent, SDS
Identical charge-to-size ratio: Amount of SDS per
unit mass of protein is constant (1.4g of SDS
binds per gram of protein)
Very power technique: Measures relative molecular
masses of polypeptide / protein
c) Agarose Gel:
Agarose, obtain from seaweeds, linear polymer of DGal and 3,6-anhydroGal
agarose dissolved in boiling water when cooled forms
a gel (held by H-bonds),
pore size relatively large: large proteins or nucleic
acids (too large to enter PAG)
Contaminants charge groups (sulphate and carboxylic
acid ) interfere with separation
Factors affecting electrophoretic mobility:
Number of parameter of electrophoretic system:
i) for zone EP, type of support medium chosen,
and if it is a gel, its pore size
ii) pH of the electrophoresis buffer
iii)ionic composition of buffer
iv) applied voltage
v) temperature
Support medium: selection is based on following
consideration
a) size of the molecules to be analyzed
b) quantity of sample available
c) cost of support medium to be used
d) availability of suitable equipment
e) purpose of the analysis
f) time to run the analysis
g) expertise of the operator
pH of the electrophoresis buffer:
DNA/RNA: Net charge negative(Phosphate group)
charge-to-size-ratio of all polynucleotide: same
Separation based on size
changing pH of electrophoretic buffer: No affect on
separation
For protein: pH value <Isoelectric point molecules
have net positive charge,and vise versa
pH of buffer solution depends upon their temperature
Ionic composition of EP buffer:
Effect on electrophoretic mobility :Charged
molecules  surrounded by an ionic atmosphere
(opposite charge)  decreased mobility
Ionic strength as low as possible to minimize counter
ion effects
Limitation : Some protein insoluble at very low salt
concentration
Applied voltage
• Increase:
-charged molecule migrate faster
-Increases current
- temperature
• Optimal value
Temperature
• Better at low temp(40C) : Otherwise density
difference  convection current Disturbed
diffusion
• Distortion of Zones:Column gel migrating
Center (warmer) faster than outer (cooler)
• Evaporation:increase in ionic strength of the
buffer
• Viscosity: agarose gels becomes softer
Detection of sample
Depends on
• the nature of the molecule to be detected
• type of electrophoresis system used
• purpose of the analysis
A)Optical methods:
1. UV absorption by separated components
Proteins: Absorb UV (230-280nm)
Limitation:Trace unpolymerized acrylamide amide
group absorb radiation
Polynucleotides absorb UV at 260nm (heterocyclic
ring resonance)
Polynucleotides : Scanned by a densitometer 
numerical readout of absorbance
Staining
• by reacting separated components with a stain or
dye and detecting by absorption or fluorescence
• stain should be - selective, react quickly, yield stable
product
• staining can be done A) before electrophoresis
(rainbow markers), B) after electrophoresis- by
immediately fixing the separated component (drying
if paper or precipitating (by fixative) if gel,)
• Destaining: excess unbound stain removed
Chromogenic
stains
Fluorescent stains
b) Radiochemical methods:
• Labeling with a radioactive isotope(before) 
Support medium cut into pieces Measured by
liquid scintillation counting/ read in
autoradiography
• high energy emitters are extremely useful labels for
autoradiography (32P)
• Protein - 125I(γ emitter) ; nucleic acid - 32P (high
energy β emitter) ; 14C and 35S are medium-energy
β emitter and 3H is a low energy β emitter – used as
tracers
Liquid Scintillation counting:
Support medium is dried, cut into strips or sections
Samples in gel support media need to be solubilized
before measurement
i) elution from the slice by an appropriate buffer
(solubiliser)
ii) Gel structure may be destroyed using sonication or
chemicals (formamide, H2O2)
Autoradiography
• Detecting radioactivity in labeled &
separated compounds
• Dried gel  Specially designed photographic
cassette (several days)  Radiation passing
through the photographic film  Reflected as
fluorescent emission (Intensifying screen) 
Enhanced image
High Voltage EP:
Separation of urinary
putrescine, cadaveiine,
histamine, spermidine,and
spermine by HVPEP
putrescine, cadaverine, histamine, spermidine, and spermine can be
completely separated by high-voltage paper electrophoreais