In this slide contains introduction, methods, supporting media for zone electrophoresis. Presented by: Mary Vishali. (Department of pharmacology), RIPER, anantapur.

1. Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Zone Electrophoresis
A Seminar as a part of curricular requirement for
Master of Pharmacy,
I Year - I semester
Presented by
Mary Vishali
(20L81S0104)
Dept of. Pharmacology
Under the guidance of
Dr. P. Ramalingam M.Pharm, Ph.D.
Professor & Director of R& D cell

2. RIPER
AUTONOMOUS
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Contents:
2
 Introduction
 Zone Electrophoresis
 Methods of Zone Electrophoresis
 Buffers
 Supporting media
 References

3. RIPER
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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Introduction
3
Definition:
 Electro means Electricity
 Phoresis means Separation
 Separation of serum proteins by the effect of an electric current.
 Electrophoresis is a physical method of analysis which involves separation of the
compounds that are capable of acquiring electric change in conducting electrodes.
 Electrophoresis may be defined as the migration of the charged particle through a
solution under the influence of an external electrical field.

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 Ions that are suspended between two electrodes tends to travel towards
the electrodes that bears opposite charges.

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 The transport of particles through a solvent by application of an electric field is
called as electrophoresis.
 Most of the polymers (containing macromolecules) are electrically charged and
will therefore move in an electric field.
 Electrophoresis is useful in identification and structure determination of such big
molecules.

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Types of
Electrophoresis
Zone
Electrophoresis
Moving
Boundary
Electrophoresis

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Zone
Electropohresis
Paper
Electropohresis
Cellulose
Acetate
Membrane
Gel
Electrophoresis
Agarose Gel
Electrophoresis
Polyacrylamide
Gel
Electrophoresis

8. RIPER
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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 8
Moving boundary
electrophoresis
Capillary
Electrophoresis
Capillary
Zone
Electrophoresis
Capillary
Gel
Electrophoresis
Micellar
Electrokinetic
Chromatography
Capillary
Isoelectric
focusing
Capillary
Isotachophoresis
Capillary
Electro
Chromatography
Isoelectric
Electrophoresis
Isotachophoresis

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Zone Electrophoresis
 In this method, an inert polymeric supporting media is used between the
electrodes to separate and analyze the sample.
 The supporting media used in zone electrophoresis are absorbent paper, gel of
starch, agar and polyacrylamide.
 The major advantage of presence of supporting media is that it minimizes mixing
of the sample and immobilization of the molecule after electrophoresis.
It makes the analysis and purification of the molecule from the gel much easier
than the moving boundary electrophoresis.

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Paper Electrophoresis
 Paper electrophoresis (PE) is useful for the separation of small-charged
molecules, such as amino acids and small proteins using a strip of paper
(chromatography paper).
 In this technique, the motion of colloidal particle of solution occurs leading to
subsequent separation along the paper strip.
 PE is easier in comparison to gel electrophoresis.
It does not require matrix preparation and it does not contain charges that interfere
with the separation of compounds.

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A strip of filter paper is moistened with buffer and the ends of the
strip are immersed into buffer reservoirs containing the electrodes.
The samples are spotted in the center of the paper and high voltage is applied.
Application of high voltage causes less diffusion of small molecules giving
better resolution and it take less time to complete the process.

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Cellulose Acetate Strip Electrophoresis
 Many biological samples adsorb on cellulose, that is paper.
The adsorption is because of hydroxyl groups present in cellulose.
Adsorption reduces the movement and therefore causes tailing of spots/bands
This spreading of spots reduces resolution.
To solve this problem cellulose acetate membrane is used where most of the
hydroxyls have been converted acetate groups.
Cellulose acetate is preferred because of its simplicity and high resolution at low
applied voltage.

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 It contains 2-3 acetyl groups per glucose unit and its adsorption capacity
is less than that of paper.
 It gives sharper bands.
 Provides a good background or staining glycoprotein

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Gel electrophoresis
 It makes the use of gel as a support matrix.
 Most popular and commonly used method.
 Used for both analytical and preparative processes.
 It is the most common method to carry out the process of electrophoresis.

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Principle:
 In this porous gel matrix is used which consist of the cross-linked polymer
network.
 Through this network, molecules of different size, charge and shape pass
through.
 This relies upon the fact that negatively charged molecule will attract towards
the positive end and vice versa.

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 Horizontal (Agarose gel electrophoresis)
 Vertical (SDS-PAGE)
17
Types of Gel Electrophoresis

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To carry out this kind of electrophoresis, following steps involve:
First, take agarose into the water to make the slurry or to dissolve the agarose.
Cool the agarose solution, and then transfer it to the casting tray containing comb.
Add electrophoresis buffer (Tris-acetate EDTA buffer) to cover the gel up to 1mm.
Then, take the DNA sample.
 Add the desired amount of 6X gel loading buffer.
 After that, take out the comb and slowly load the sample mixture containing
tracking dye (Bromophenol blue) into the wells through the micropipette.

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Close the lid of the gel tank, and turn on the power supply so that DNA
can migrate towards the positive electrode.
At last, remove the gel tray and place is in a UV transilluminator, to see the
orange-red coloured DNA bands.

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SDS-PAGE
 It stands for Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis and
includes the following steps:
 First, add the resolving gel between the two glass plates of the casting frame.
 Then, place a comb on the glass plates leaving 1cm space.
 Add isopropanol on the top of the gel.
After solidification of resolving gel, remove the isopropanol using filter paper.

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 Load the stacking gel on the top of the glass plates.
 Then, place a comb and solidify the stacking gel for desired time.
 After solidification of stacking gel, remove the comb that forms a well.
 Place a ladder into the extreme right and place the protein sample with a tracking
dye (Bromophenol blue) into the other wells, by using a micropipette.
 Then turn on the voltage supply, so that tracking dye can cross the gel by forming
different bands.
 After the completion of electrophoresis, take out the gel and rinse it with deionized
water 4-5 times to remove SDS and buffer.

23. RIPER
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 Then, dip the gel in the Coomassie blue stain which is a staining
buffer, stains the invisible protein bands after a few hours.
23

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Capillary Zone Electrophoresis (CZE)
 CZE analytes move in the EOF but separate into bands because of differences in
their electrophoretic mobilities, µ. Differences in µ make each analyte's overall
migration velocity slightly different, and difference in migration velocity =
separation. µ's are roughly a function of analyte charge and frictional and size
differences.
 In the adjacent image, three peaks are traveling down the capillary from the
beginning of the capillary on the left to the detector and exit reservoir on the right
(again, this is a simple schematic).

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 In this system, an absorption detector would work if the
analytes have good molar absorption at wavelengths the
detector has available (usually in the UV). If the analytes are poor
absorbers then a strong UV absorber can be added to the run buffer
and the decrease in absorption—when the analytes pass the detector
can be used to detect the analytes. This last is called indirect
absorption.
25

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 All that's required in the CZE method is a well-chosen buffer
in the initial buffer reservoir.
• Separation occurs because of relatively simple interaction of the
analytes with the pH of the buffer.
• This technique is also called free solution capillary electrophoresis
(FSCE) for that reason.
• The capillary is often pre-washed with the buffer; the sample—
dissolved in the same buffer—is injected; EOF is established; and you're
off.
26

27. RIPER
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• This should be contrasted with the methods discussed below.
• In the adjacent figure, two of the peaks have already passed
the detector and the third is about to.
27

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29. RIPER
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Buffers
 Biological buffers are an essential part of electrophoresis techniques. They
usually added to solution in order to make the pH stable while a current is
carried through the sample.
 Additionally buffers provide the ions necessary for electrophoresis migration.
 The ideal buffer for electrophoresis depends on the isoelectric point (the pH at
which a particular molecules carriers no net electrical charge) of the sample
being analyzed.
29

30. RIPER
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 Although pre-made buffers solutions for electrophoresis are available
in the market.
30

31. RIPER
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Examples
 Tris buffer
 Mops buffer
 Bis – Tri buffer
 ACES buffer
 Bicine buffer
 CAPS
 CAPSO
 CHES
 Tricine buffer
31

32. RIPER
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Supporting media
 The supporting media used zone electrophoresis are absorbent paper, gel of
starch, agar and polyacrylamide.
 Major advantage of presence of supporting media is that it minimizes mixing of
the sample and immobilization of the molecule after electrophoresis
32

33. RIPER
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Supporting media examples
 Ethidium bromide
 Coomassie blue staining
 Silver staining
 Zinc staining
 Fluorescent staining
 Glycoprotein staining
 Heme protein staining
33

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 After electrophoresis the molecules in the gel can be stained to make them
visible.
 After electrophoresis the movement of sample molecules can be visualized
by adding stains.
34

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References
 Mikkers FE, Everaerts FM, Verheggen TP. High-performance zone
electrophoresis. Journal of Chromatography A. 1979 Feb 1;169:11-20.
 Mikkers FE, Everaerts FM, Verheggen TP. Concentration distributions in free zone
electrophoresis. Journal of Chromatography A. 1979 Feb 1;169:1-0.
 Shihabi ZK. Stacking in capillary zone electrophoresis. Journal of
Chromatography A. 2000 Nov 24;902(1):107-17.

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Thank You