2. CONTENTS
INTRODUCTION
THEORY
TYPES OF ELECTROPHORESIS
MOVING BOUNDARY ELECTROPHORESIS
ZONE ELECTROPHORESIS
PRINCIPLE AND TYPES OF ZONE ELCTROPHORESIS
GEL ELECTROPHORESIS
PRINCIPLE OF GEL ELECTROPHORESIS
AGAROSE GEL PREPRATION AND INSTRUMENTATION
SAMPLE PREPRATION
GEL LOADING AND MICROPIPETTING TECHNIQUE
GEL STAINING
POLY ACRYLAMIDE GEL ELECTROPHORESIS (PAGE)
PREPRATION OF PAG AND ITS COMPONENTS
COMPARISION OF FLATBED SYSTEM AND VERTICAL GEL
SYSTEM
ISO-ELECTRIC FOCUSSING
SDS-PAGE
4. ELECTROPHORESIS
Electrophoresis is also called Cataphoresis.
Electrophoresis is a separation technique in which ions
in colloidal solutions are separated based upon their
differences in size and charge when a high voltage is
applied to the solution[1]
Positive ions migrate to the negative electrode
(cathode), and negative ions migrate towards the positive
electrode (anode).
Was developed into workable system by Arne Tiselius of
Sweden in 1930. He won Nobel prize for the work in 1948.
Introduction:
5. Theory of electrophoresis
Mobility- rate of movement ,denoted by “μ”.
Particle to migrate in electric field requires a net electrostatic charge.
Consider a particle that is placed in a container of liquid, saturated
with the buffer and has a potential applied to it. The force “F” is equal
to:
F= QE Where,
Q= charge of the particle
E= field strength
As particle moves in the buffer it meets retardation force caused by
the viscosity of the solvent which can be expressed as:
Fs= 6πrημ Where,
fs = viscous retardation force
r = radius of particle in cm
η = viscosity of mediun in poise
μ = electrophoretic velocity in cm/sec
Now when both the forces are equal i.e
Fs = QE = 6πrημ
6. Hence mobility of particle can be defined as:
μ = υ/E where,
μ = mobility in cm2
volt-1
sec-1
E = volt cm-1
After substitution,
μ = Q/6πrη
Factor affecting the mobility[1]
Heat production
Smiling
Electro-osmosis correction
7. Moving boundary electrophoresis :
Density gradient electrophoresis
Iso-tachophoresis
Zone electrophoresis:
Paper electrophoresis
Gel electrophoresis
Capillary zone electrophoresis
Immuno- electrophoresis
Iso-electric focusing electrophoresis
Types of electrophoresis
8. Separation occurs due to difference
in mobility of molecules. Mobility is
proportional to m/e ratio.
The position of moving ions, which
forms a boundary, which is detected
by measuring the changes in
refractive index throughout solution.
The concentration gradients which
are formed during electrophoresis are
usually detected by optical method.
Moving boundary electrophoresis[2]
This method allows the charged species to migrate in a free moving
solution in absence of a supporting medium.
Samples are fractioned in a U shaped tube that has been filled with
unstabilized buffer.
An electrical field is applied by means of electrodes at the ends of
the tube.
Fig1: illustration of moving
boundary electrophoresis
9. Advantages:
1.Application to a wide variety of high molecular weight
substances.
Disadvantages [3]
1.Mixing of separated compound as a consequence of
thermal and density gradient as well as mechanical vibration.
2.Thermal vibration and mechanical vibration controlling is
difficult and expensive.
3.Detection of fraction separated with optical system also
adds to the expenses.
ADVANTAGE AND DISADVANTAGES OF MOVING
BOUNDARY ELECTROPHORESIS
10. ZONE ELECTROPHORESIS
Any electrophoretic technique in which components are
separated into zones or bands in a buffer and stabilized in
solid, porous, or any other support medium e.g.: paper strip,
agar gel or poly-acrylamide gel.
Separates macromolecular colloids e.g.. proteins in serum,
urine, Cerebrospinal fluids (CSF), erythrocytes; nucleic acids.
Types of zone electrophoresis are:-
1. Paper electrophoresis
2. Gel electrophoresis
3. Capillary zone electrophoresis
4. Immuno- electrophoresis
11. ADVANTAGES AND DISADVANTAGES OF ZONE
ELECTROPHORESIS
ADVANTAGES:
1. Useful in biochemical investigation.
2. Very small quantity of samples can be analyzed.
3. Useful to study both simple and complex mixtures equally.
4. Equipment cost is low and maintenance is easy.
5. Detection and visualization with various reagents and dyes
are possible
6. Permits 2-D electrophoresis for higher resolution.
7. Quantification by densitometry and auto-radiography can
be done
DISADVANTAGE:
1. Unsuitable for accurate mobility and iso-electric point
determination.
2. Complications such as capillary flow, electro osmosis,
adsorption and molecular sieving are introduced.
12. GEL ELECTROPHORESIS[4]
Gel electrophoresis is a widely used technique for the
analysis of nucleic acids and proteins. 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.
Types of gel electrophoresis:-
1. One-dimensional - Agarose gel electrophoresis, Poly-
acrylamide gel electrophoresis (native or SDS-PAGE) and Iso-
electricfoccusing (IEF) or
2. Two dimensional - 2D-PAGE.
3. Capillary electrophoresis
13. +-
Power
DNA
PRINCIPLE OF GEL ELECTROPHORESIS
DNA is negatively charged
When placed in an electric field, DNA will migrate towards the
positive pole (anode).
An agarose gel is used to slow the movement of DNA and
separate by size.
14. Fig 2 :Scanning Electron Micrograph of Agarose Gel (1×1 µm)
• Polymerized agarose is porous, allowing for the movement of
DNA
15. +-
Power
DNA
How fast will the DNA migrate?
• Strength of the electrical field, buffer, density of agarose gel
• Size of the DNA
• Small DNA move faster than large DNA
• Gel electrophoresis separates DNA according to size
small
large
Within an agarose gel, linear DNA migrate inversely
proportional to the log10 of their molecular weight.
16. AGAROSE [5]
•Agarose is a linear polymer
extracted from seaweed.
• 0.7% - for large DNA (5-10
kb)
• 2%- for small DNA (0.2-
1kb)
• Recommended
concentration is 1%
•Agarose was first used in
biology when Robert Koch
used it as a culture medium
for Tuberculosis bacteria in
1882
Fig 3:- Agarose used for
electrophoresis
18. An agarose gel is prepared by
combining agarose powder and a
buffer solution.
Agarose
Buffer
Flask for boiling
Buffer used- Tris-borate buffer (pH
8.0) that contains EDTA (TBE);
EDTA is a chelating agent that
binds divalent cations such as
Mg++ that many nucleases
require for their activity; EDTA
thus protects the DNA from
enzymatic degradation[6]
Fig: 4
19. Casting tray
Gel combs
Power supply
Gel tank
Cover
Electrical leads
Electrophoresis Equipment
Fig 5: Electrophoresis Equipment
20. Gel casting tray & combs
Fig :6
Small 8x10 cm gels (minigels) are very popular.
The volume of agarose required for a minigel is around 30–50 mL,
for a larger gel it may be 250 mL.
21. Seal the edges of the casting tray and put in the combs. Place the casting
tray on a level surface. None of the gel combs should be touching the
surface of the casting tray.
Preparing the Casting Tray
Fig: 7
22. Fig 8:Agarose Fig: 9 Buffer Solution
Combine the agarose powder and buffer solution. Use a flask
that is several times larger than the volume of buffer.
23. Fig 10: Agarose is insoluble at
room temperature.
Gently swirl the solution periodically when heating to allow all the grains of
agarose to dissolve.
Be careful when boiling - the agarose solution may become superheated
and may boil violently if it has been heated too long in a microwave oven.
Melting the Agarose
Fig 11: The agarose solution is
boiled until clear.
24. Allow the agarose solution to cool slightly (~60ºC) and then
carefully pour the melted agarose solution into the casting tray.
Avoid air bubbles.
Pouring the gel
Fig :12
25. Each of the gel combs should be submerged in the melted agarose
solution.
Fig : 13
26. When cooled, the agarose polymerizes, forming a flexible gel. It
should appear lighter in color when completely cooled (30-45
minutes). Carefully remove the combs and tape.
Fig : 14
27. Place the gel in the electrophoresis chamber.
Fig: 15
28. buffer
Add enough electrophoresis buffer to cover the gel to a
depth of at least 1 mm. Make sure each well is filled with
buffer.
Cathode
(negative)
Anode
(positive)
wells
DNA
Fig: 16
29. 6X Loading Buffer:
• Bromophenol Blue (for color)
• Glycerol (for weight)
Sample Preparation
Mix the samples of DNA with the 6X sample loading buffer (w/
tracking dye). This allows the samples to be seen when loading
onto the gel, and increases the density of the samples, causing
them to sink into the gel wells.
Fig 17: prepared DNA sample
30. Loading the Gel
Carefully place the pipette tip over a well and gently expel the
sample. The sample should sink into the well. Be careful not to
puncture the gel with the pipette tip.
Fig 18
34. Place the cover on the electrophoresis chamber, connecting the
electrical leads. Connect the electrical leads to the power supply. Be
sure the leads are attached correctly - DNA migrates toward the
anode (red). When the power is turned on, bubbles should form on
the electrodes in the electrophoresis chamber.
The electrophoresis is run by 70-100 V/20-80 mA for about an hour or at
20 to 30 V overnight [6]
RUNNING THE GEL
Fig 22 Fig 23
36. 100
200
300
1,650
1,000
500
850
650
400
12,000 bp
5,000
2,000
DNA LADDER STANDARD
Inclusion of a DNA ladder (DNAs of know sizes) on the gel
makes it easy to determine the sizes of unknown DNAs.
-
+
DNA
migration
bromophenol blue
37. STAINING THE GEL
***CAUTION! Ethidium bromide is a powerful mutagen and is moderately
toxic [7]
• Ethidium bromide binds to DNA and fluoresces under UV light,
allowing the visualization of DNA on a Gel.
• Ethidium bromide can be added to the gel and/or running
buffer before the gel is run or the gel can be stained after it has
run.
38. SAFER ALTERNATIVES TO ETHIDIUM BROMIDE
Methylene Blue
Carolina BLU Stain
EVA green [8]
Advantages
Inexpensive
Less toxic
No UV light required
No hazardous waste disposal
Disadvantages
Less sensitive
More DNA needed on gel
Longer staining/destaining time
stains Compounds
Amido black 10 B Proteins
Coomassie blue Proteins
Sudan black Lipid & lipoproteins
Ninhydrine Amino acid
Other stains which are used [1]
39. STAINING THE GEL
• Place the gel in the staining tray containing warm diluted stain.
• Allow the gel to stain for 25-30 minutes.
• To remove excess stain, allow the gel to destain in water.
• Replace water several times for efficient destain.
Fig 27
40. Ethidium Bromide requires an ultraviolet light source to visualize
DNA bands
under UV light in
an ethidium-
bromide-stained
gel is easily
visible if it
contains about
20 ng of DNA.
Fig 28
41. POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE)
PAGE, is one of the most widely used electrophoresis techniques
and separates proteins through a polyacrylamide gel matrix[4]
Two types of PAGE can be carried out
1.Native PAGE- in which electrophoresis is carried out under non-
denaturing conditions and separation is based on the protein’s
charge and hydrodynamic size or
2. SDS PAGE - in which proteins are denatured prior to
electrophoresis and separation is based on a protein’s mass or
molecular weight.
The chemical SDS (sodium dodecyl sulphate) is an anionic
detergent which, in combination with DTT (dithiothreitol) or β-
mercaptoethanol, breaks intramolecular bonds in the protein
destroying any secondary, tertiary or quaternary structure.
This leaves only the linear primary amino acid structure of the
protein which will contain an overall negative charge proportional to
its mass, therefore allowing the proteins to be separated solely on
the basis of their molecular mass.
42. The gel typically consist of acrylamide, bisacrylamide, SDS, and a
buffer with an adjusted pH.
The solution may be degassed under a vacuum to prevent the
formation of air bubbles during polymerization.
A source of free radicals and a stabilizer such as ammonium
persulfate and TEMED are added to initiate polymerization.
The polymerization reaction results in a gel because of the added
bisacrylamide, generally about 1 part in 35 relative to acrylamide,
which can form cross-links between two polyacrylamide molecules.
The ratio of acrylamide to bisacrylamide can be varied for special
purposes.
The acrylamide concentration of the gel can also be varied,
generally in the range from 5% to 25%.
Lower percentage gels are better for resolving very high molecular
weight proteins, and viceversa.
PREPARING POLY-ACRYLAMIDE GELS[9]
43. COMPONENTS OF POLYACRYLAMIDE GEL ARE [9]
:-
1.Chemical buffer -to stabilizes the pH value to the desired value-
Tris , Bis-Tris or imidazole.
2.Counterion -to balance the intrinsic charge of the buffer ion and
also affect the electric field strength during electrophoresis – glycine
and tricine.
3.Acrylamide.
4.Bisacrylamide - used as cross linking agent .
5.Sodium Dodecyl Sulfate - denature native proteins
6.Ammonium persulfate - as an initiator for gel formation.
7.TEMED (N, N, N', N'-tetramethylethylenediamine)- stabilizes
free radicals and improves polymerization.
44. CHEMICALS FOR PROCESSING AND VISUALIZATION
Tracking dye:- Anionic dyes of a known electrophoresis mobility are
used like Bromophenol blue to follow colorless protein .
Being a highly mobile molecule it moves ahead of most proteins. As it
reaches the anodic end of the electrophoresis medium electrophoresis
is stopped.
Loading aids:- To ensure that the sample sinks to the bottom of the
gel glycerol and sucrose are used to increase the density of the
sample.
Coomassie Brilliant Blue R-250 (CBB) - is an anionic dye.
Proteins in the gel are fixed by acetic acid and simultaneously stained.
The excess dye incorporated into the gel can be removed by
distaining with the same solution without the dye. The proteins are
detected as blue bands on a clear background
46. ADVANTAGES OF PAGE ELECTROPHORESIS
1. Excellent separation on basis of size, shape, and charge
2. Pore size and the amount of cross linking can be controlled
3. Excellent resolution
4. Separation is rapid (30 min to a few hours)
5. Apparatus is relatively simple to operate
6. PAGE has a high loading capacity, up to 10 micrograms of
DNA can be loaded into a single well (1 cm x 1 mm) without
significant loss of resolution.
7. PAGE is an ideal gel system from which to isolate DNA
fragments for subcloning and other molecular biological
techniques.
47. As any other methods, PAGE also has disadvantages:
1. The mobility of the fragments can be affected by base
composition making accurate sizing of bands a problem.
2. Polyacrylamide quenches fluorescence, making bands
containing less than 25 ng difficult to visualize with
ethidium bromide staining.
3. Quantification difficult by itself - Increased when combined
with procedures using radiolabeled or photometric
markers
4. Acrylamide is a neurotoxin
48. Flatbed Systems: Vertical Systems:
Gel thickness is limited, because
cooling is only possible from one side
Higher protein loading capacity,
because thicker gels can be used,
which are cooled from both sides
Blotting is easier because of higher
gel thickness
One gel per instrument is run Multiple gel runs possible
Very versatile for different methods,
ideal for isoelectric focusing
Limited technical possibilities, not
optimal for isoelectric focusing
Thin layers can easily be used,
easy sample application
The thinner the gel, the more
complicated is sample application
Easy to handle and to clean,
no glass plates necessary, thus ideal
for routine applications
Many pieces to set up and to clean
COMPARISON OF FLATBED AND VERTICAL GEL SYSTEMS[4]
49. ISO ELECTRIC FOCUSSING[1] [4]
It is mainly used for a separation of electrolytes such as proteins.
When electrophoresis is run in solution buffered at constant pH,
proteins having net charge will migrate towards opposite electrode.
The use of pH gradient across supporting medium causes each protein
to migrate to an area of specific pH.
A sharp well defined protein bands occur at the point where iso electric
point equals to pH of gradient
Separation is carried out on gels on which a stable a pH gradient has
been established.
An ampholytic compound has a pH at which it is neutral.
The pH gradient is achieved by impregnating the gel with polyamino-
polycarboxylic acids.
When subjected to electric field these migrate and come to rest in
order of their pH.
Thus each ampholyte migrates in applied field until it reaches a
position on the plate where pH of medium is equal to iso electric point.
50. At this point ampholyte is in its zwitter ion form and is
neutral.
Thus it losses electrophoretic mobility and becomes focused
in narrow zone at this point.
Fig 31: illustrating IEF
51. Advantages:
1.Spreading of bands is minimized.
2.Proteins that differ by little as 0.01pH can be adequately resolved
Disadvantages:
1.As carrier ampholytes are used in high concentration, a high voltage
power supply is necessary. As a result the electrophoretsis may be
affected.
ADVANTAGES AND DISADVANTAGES OF IEF
52. SDS-PAGE (PolyAcrylamide Gel Electrophoresis)[9]
SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel
electrophoresis, is a technique widely used in biochemistry, forensics,
genetics and molecular biology:
1.To separate proteins according to their electrophoretic mobility (a
function of length of polypeptide chain or molecular weight).
2.To separate proteins according to their size, and no other physical
feature.
SDS (sodium dodecyl sulfate) is a detergent (soap) that can
dissolve hydrophobic molecules but also has a negative charge
(sulfATE) attached to it.
SDS (the detergent soap) breaks up hydrophobic areas and coats
proteins with negative charges thus overwhelming positive charges in
the protein.
The detergent binds to hydrophobic regions in a constant ratio of
about 1.4 g of SDS per gram of protein.
Therefore, if a cell is incubated with SDS, the membranes will be
dissolved, all the proteins will be solublised by the detergent and all
the proteins will be covered with many negative charges
53. Fig 32 : illustrating the effect of SDS over protein moleculesfi
54. COMPONENTS OF SDS PAGE GEL[9]
Running Buffer: Tris/Glycine:
Glycine(pKa=9.69) is a trailing
ion (or slow ion). In other
words it runs through the gel
slower then the slowest protein
at a pH above 8.0.
Stacking Gel: Is prepared w/Tris/HCL buffer pH 6.8, ~2pH units
lower than running buffer. Large pore polyacrylamide used to align
and create a thin starting zone of the protein of apx. 19µm on top of
the resolving gel.
Resolving Gel: Small pore polyacrylamide gel (3 - 30% acrylamide
monomer) typically made using a pH 8.8 Tris/HCl buffer. Resolves
protein ~24 – 205 kDa
Fig: 32
55. TWO DIMENSIONAL ELECTROPHORESIS
Separation of hundreds of proteins
based on:
Isoelectric point
Molecular weight
Popular method for protein display
and proteomics-one spot at a time
Permits simultaneous detection,
display, purification, identification,
quantification, pI, and MW.
Robust, reproducible, simple, cost
effective, scalable
Provides differential quantification
using Differential 2D Gel
Electrophoresis (DIGE)
Fig33: The principle of the 2-D electrophoresis according to
O’Farrell (1975).
56. PROCESSES INVOLVED IN 2D GEL ELECTROPHORESIS
Protein isolation and quantification
Isoelectric focusing (first dimension)
SDS-PAGE (second dimension)
Visualization of proteins spots with Dye
Identification of protein spots with Mass Spec
57. CAPILLARY ELECTROPHORESIS[1]
Also known as:
High performance capillary electrophoresis
Capillary zone electrophoresis
Free solution capillary electrophoresis
Capillary electrophoresis is a micro-electrophoretic system in
which separation takes place in a 10-100μm internal diameter,
fused quartz, hollow capillary tube from 30-100 cm long with
each end immersed in a buffer.
DC applied upto 300V/cm.
WHY CAPILLARY ELECTROPHORESIS?
Reduces the problem resulting from heating effect- more
surface area to volume ratio hence more heat dessipation.
Reduce zone broadening.
58. Forces associated with the capillary electrophoresis are
Electro-osmotic flow (EOF)
Electro-phoretic separation
Under these influence all components in the sample travels in
one direction towards cathode.
The electro-osmotic flow (EOF) is caused by applying high-
voltage to an electrolyte-filled capillary.
This flow occurs when the buffer running through the silica
capillary has a pH greater than 3 and the SiOH groups lose a
proton to become SiO-
ions.
The capillary wall then has a negative charge, which develops
a double layer of cations attracted to it.
The inner cation layer is stationary, while the outer layer is
free to move along the capillary.
The applied electric field causes the free cations to move
toward the cathode creating a powerful bulk flow.
in CE potential applied is greater then normal electrophoresis
i.e 300V/cm this facilitates electro-phoretic separation.
PRINCIPLE OF CAPILLARY ELECTROPHORESIS
60. COMPONENTS OF CE
1. COLUMN
2. SAMPLE
3. BUFFERS
4. MODIFIERS
5. POWER SUPPLY
6. DETECTORS
COLUMNS:- borosilicate or fused quartz
Dimensions : internal diameter 10-100μm
length 30-100cm
capillary wall 300-600 μm
SAMPLE:- 5-50nL
Added by placing one end of the capillary into sample
containers raised to a set height (10cm) for a measured
time (30sec) and allowing a siphoning action takes place
or place one end into the sample and apply a potential for
a short time.
61. BUFFERS: 0.5mM concentration is common
10mM mannitol
0.05M pH 7 phosphate, borate etc
MODIFIERS:- used if EOF is not demanded.
these alter the direction and rate of EOF
reverse flow- cetyltrimethyammoniunbromide
tetradecyltrimethylammonium bromide
zero flow-s-benzylthiouronium chloride
reduce flow- methanol
increase flow- acetonitrile
POWER SUPPLY:- faster heat dessipation hence high
potential of 300V/cm can be applied.
DETECTORS:- U-V-visible detectors, fluorescence
detectors, mass spectrometry,radioisotopes,
conductometry and amperometry etc
62. 1. CE has a flat flow, compared to the pumped parabolic flow of the
HPLC. The flat flow results in narrower peaks and better resolution.
2. CE has a greater peak capacity when compared to HPLC—CE
uses millions of theoretical plates.
3. HPLC is more thoroughly developed and has many mobile and
stationary phases that can be implemented.
4. HPLC has more complex instrumentation, while CE is simpler for
the operator.
5. HPLC has such a wide variety of column lengths and packing,
whereas CE is limited to thin capillaries.
6. CE require less sample volume then HPLC.
Capillary Electrophoresis versus High Performance Liquid
Chromatography (HPLC) [10]
CE HPLC
Length of column 30-100cm 3-25 cm,25-
50cm(microbore)
Internal diameter 10-100μm 4-6 mm,1-2 mm(microbore
Sample volume 5-50 nL 20μL
63. COMPARISION OF CLASSICAL GEL AND CE
Classical gel electrophoresis Capillary electrophoresis
Gels – polyacrylamide or agarose,
Slabs length and width 5-25 cm
Columns length- 7-10cm,internal
diameter- 5mm
Fused quartz capillary
Length- 30-100cm, internal diameter-10-
100μm
Electrophoretic separation Electrophoretic and electro-osmotic
Applied field100-2kV Applied field 10-50 kV
Heat dessipation is slow from column
and quicker in slabs
Heat dessipation is rapid
0.05-0.5M electrolyte for conductivity
and stability
Sample volume 1-50 μL
Different sample can be analysed
Same as classical gel
Sample volume 1-50nL
Only one sample analysed at a time
Chromogenic agent or staining agent for
detection of solute
HPLC detectors ,U-V absorbance and
fluorescence are commonly used
Slow, limited resolution and time
consuming.
High resolution, efficiency and
sensitivity, is modrately fast
64. ADVANTAGES AND DISADVANTAGES OF CE
Advantages
Offers new selectivity, an alternative to HPLC
Easy and predictable selectivity
High separation efficiency (105
to 106
theoretical plates)
Small sample sizes (1-10 ul)
Fast separations (1 to 45 min)
Can be automated
Disadvantages
Cannot do preparative scale separations
Reproducibility problems
65. REFRENCES
1. Milons
2. Elctrophoresis in practice, fourth edition Reiver Westermeir
3. Instrumental method of chemical analysis. By B.K.Sharma, page
no- 269
4. Randox Research & Development catalogue 2009/10
5. HHMI undergraduate research studio – Freshman, Biology
section, Fall 2007 – Agarose Gel Electrophoresis.
6. www.wikipedia.org/wiki/Gel_Electrophoresis.
7. www.wikipedia.org/wiki/ethidium_bromide
8. Biotech.about.com/../DNA strain.htm
9. www.wikipedia.org/wiki/SDS_PAGE
10. http://elchem.kaistac.kr/chem-ed
