In this slide contains introduction, methods, supporting media for zone electrophoresis.
Presented by: Mary Vishali. (Department of pharmacology),
RIPER, anantapur.
In this slide contains types, working principle, factors affecting, advantage and disadvantage of paper electrophoresis.
Presented by: G.Sai Swetha. (Department of pharmacology),
RIPER, anantapur.
The technique of paper electrophoresis is simple and inexpensive and requires only micro quantities of plasma for separation.
The support medium is a filter paper
The electrophoresis apparatus in its simplest form consists of two troughs to contain buffer solution, through which electric current is passed.
Frequently used in isolating proteins, amino acids and oligopeptides.
In this slide contains types, working principle, factors affecting, advantage and disadvantage of paper electrophoresis.
Presented by: G.Sai Swetha. (Department of pharmacology),
RIPER, anantapur.
The technique of paper electrophoresis is simple and inexpensive and requires only micro quantities of plasma for separation.
The support medium is a filter paper
The electrophoresis apparatus in its simplest form consists of two troughs to contain buffer solution, through which electric current is passed.
Frequently used in isolating proteins, amino acids and oligopeptides.
ION EXCHANGE CHROMATOGRAPHY
ByM.Vharshini
B.Sc. Bio Medical Science
Sri Ramachandra University
ION EXCHANGE CHROMATOGRAPHY
Ion-exchange chromatography is a process that allows the separation of ions and polar molecules based on their affinity to the ion exchanger.
It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids.
Cations or Anions can be separated using this method.
PRINCIPLE
It is based on the reversible electrostatic interaction of ions with the separation matrix (i.e.)
The separation occurs by reversible exchange of ions between the ions present in the solution and those present in the ion exchange resin.
CLASSIFICATION OF RESINS
According to the chemical nature they classified as-
1. Strong cation exchange resin
2. Weak cation exchange resin
3. Strong anion exchange resin
4. Weak anion exchange resin
According to the Source they can -
Natural resins : Cation - Zeolytes, Clay
Anion - Dolomite
Synthetic resins: Inorganic & Organic resins
◘Organic resins are polymeric resin matrix.
The resin composed of –
Polystyrene (sites for exchangeable functional groups)
Divinyl benzene(Cross linking agent)-offers stability.
Ion exchange resin should have following requirements
»It must be chemically stable.
»It should be insoluble in common solvents.
» It should have a sufficient degree of cross linking.
»The swollen resin must be denser than water.
»It must contain sufficient no. of ion exchange groups.
Physical properties of ion exchange resins
Cross linking:
It affects swelling & strength & solubility
Swelling:
When resin swells, polymer chain spreads apart
Polar solvents → swelling
Non-polar solvents → contraction
Swelling also affected electrolyte concentration.
Particle size and porosity
Increase in surface area & decrease in particle size will increase the rate of ion exchange.
Regeneration
Cation exchange resin are regenerated by treatment with acid, then washing with water.
Anion exchange resin are regenerated by treatment with NaOH, then washing with water until neutral.
EXPERIMENTAL SETUP OF ION EXCHANGE CHROMATOGRAPHY
Metrohm 850 Ion chromatography system
Instrumentation of ion exchange chromatography
PRACTICAL REQUIREMENTS
1.Column
» glass, stainless steel or polymers
2.Packing the column
» Wet packing method:
A slurry is prepared of the eluent with the stationary phase powder and then carefully poured into the column. Care must be taken to avoid air bubbles.
3.Application of the sample
After packing, sample is added to the top of the stationary phase, use syringe or pipette.
This layer is usually topped with a small layer of sand or with cotton or glass wool to protect the shape of the organic layer from the velocity of newly added eluent.
4.Mobile phase
Acids, alkalis, buffers…
6.Stationary phase
The ionic
spectrofluorometer is the instrument for recording fluorescence emission and absorption spectra When a beam of light is incident on certain substances they emit visible light or radiations. This is known as fluorescence. Fluorescence starts immediately after the absorption of light and stops as soon as the incident light is cut off. The substances showing this phenomenon are known as flourescent substances.
Isoelectric focusing electrophoresis- Principle , procedure and applicationsJaskiranKaur72
IEF separates amphoteric compounds, such as proteins, with increased resolution in a medium possessing a stable pH gradient. The protein becomes “focused” at a point on the gel as it migrates to a zone where the pH of the gel matches the protein's pI. At this point, the charge of the protein becomes zero and its migration ceases.
Electrophoresis is the movement of charged particles through an electrode when subjected to an electric Field
Cations move towards cathode
Anions move towards anode
By this technique solutes are separated by their different rates of travel through an electric field.
Commonly used in biological analysis, particularly in the separations of proteins, peptides and nucleic acids
In this slide contains principle, types, materials used, factors affecting gel electrophoresis.
Presented by: I. Sai Reddemma (Department of pharmacology).
RIPER, anantapur.
In this slide contains introduction, principle, methods, factors, application and disadvantage of Horizontal Electrophoresis.
Presented by: A.Geethanjali (Department of pharmacology),
RIPER, anantapur.
ION EXCHANGE CHROMATOGRAPHY
ByM.Vharshini
B.Sc. Bio Medical Science
Sri Ramachandra University
ION EXCHANGE CHROMATOGRAPHY
Ion-exchange chromatography is a process that allows the separation of ions and polar molecules based on their affinity to the ion exchanger.
It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids.
Cations or Anions can be separated using this method.
PRINCIPLE
It is based on the reversible electrostatic interaction of ions with the separation matrix (i.e.)
The separation occurs by reversible exchange of ions between the ions present in the solution and those present in the ion exchange resin.
CLASSIFICATION OF RESINS
According to the chemical nature they classified as-
1. Strong cation exchange resin
2. Weak cation exchange resin
3. Strong anion exchange resin
4. Weak anion exchange resin
According to the Source they can -
Natural resins : Cation - Zeolytes, Clay
Anion - Dolomite
Synthetic resins: Inorganic & Organic resins
◘Organic resins are polymeric resin matrix.
The resin composed of –
Polystyrene (sites for exchangeable functional groups)
Divinyl benzene(Cross linking agent)-offers stability.
Ion exchange resin should have following requirements
»It must be chemically stable.
»It should be insoluble in common solvents.
» It should have a sufficient degree of cross linking.
»The swollen resin must be denser than water.
»It must contain sufficient no. of ion exchange groups.
Physical properties of ion exchange resins
Cross linking:
It affects swelling & strength & solubility
Swelling:
When resin swells, polymer chain spreads apart
Polar solvents → swelling
Non-polar solvents → contraction
Swelling also affected electrolyte concentration.
Particle size and porosity
Increase in surface area & decrease in particle size will increase the rate of ion exchange.
Regeneration
Cation exchange resin are regenerated by treatment with acid, then washing with water.
Anion exchange resin are regenerated by treatment with NaOH, then washing with water until neutral.
EXPERIMENTAL SETUP OF ION EXCHANGE CHROMATOGRAPHY
Metrohm 850 Ion chromatography system
Instrumentation of ion exchange chromatography
PRACTICAL REQUIREMENTS
1.Column
» glass, stainless steel or polymers
2.Packing the column
» Wet packing method:
A slurry is prepared of the eluent with the stationary phase powder and then carefully poured into the column. Care must be taken to avoid air bubbles.
3.Application of the sample
After packing, sample is added to the top of the stationary phase, use syringe or pipette.
This layer is usually topped with a small layer of sand or with cotton or glass wool to protect the shape of the organic layer from the velocity of newly added eluent.
4.Mobile phase
Acids, alkalis, buffers…
6.Stationary phase
The ionic
spectrofluorometer is the instrument for recording fluorescence emission and absorption spectra When a beam of light is incident on certain substances they emit visible light or radiations. This is known as fluorescence. Fluorescence starts immediately after the absorption of light and stops as soon as the incident light is cut off. The substances showing this phenomenon are known as flourescent substances.
Isoelectric focusing electrophoresis- Principle , procedure and applicationsJaskiranKaur72
IEF separates amphoteric compounds, such as proteins, with increased resolution in a medium possessing a stable pH gradient. The protein becomes “focused” at a point on the gel as it migrates to a zone where the pH of the gel matches the protein's pI. At this point, the charge of the protein becomes zero and its migration ceases.
Electrophoresis is the movement of charged particles through an electrode when subjected to an electric Field
Cations move towards cathode
Anions move towards anode
By this technique solutes are separated by their different rates of travel through an electric field.
Commonly used in biological analysis, particularly in the separations of proteins, peptides and nucleic acids
In this slide contains principle, types, materials used, factors affecting gel electrophoresis.
Presented by: I. Sai Reddemma (Department of pharmacology).
RIPER, anantapur.
In this slide contains introduction, principle, methods, factors, application and disadvantage of Horizontal Electrophoresis.
Presented by: A.Geethanjali (Department of pharmacology),
RIPER, anantapur.
In this slide contains principle, types, methods and application of Western Blotting Technique.
Presented by: T.NIRANJAN REDDY (Department of pharmacology).
RIPER, anantapur
In this slide contains Interference In Atomic Absorption Spectroscopy and applications.
Presented by: Shaik Gouse ul azam. ( department of pharmaceutical analysis.)
RIPER, anantpur.
In this slide contains introduction, principle, application, advantage and disadvantage of Vertical Gel Electrophoresis
Presented by: Shaik Firdous Banu. (Department of pharmacology),
RIPER, anantapur.
Introduction to Analytical Techniques in Phaese III,
Spectrophotometry, Reflectance photometry, Nephelometry & Turbidimetry, Osmometry, Potentiometry, Flowcytometry, Densitometry, Electrophoresis, LC-MS, ICP-MS
Presented by
B. Kranthi Kumar
Department of Pharmacology
In this slide contains analytical techniques in phase-3 clinical trials.
Presented by: KRANTHI KUMAR BONALA (Department of pharmacology).
RIPER, anantapur
In this slide contains deep explanation about Ionization Techniques in LC-MS.
Presented by: G Chiranjeevi. (Department of pharmaceutical analysis)
RIPER, anantpur.
In this slide contains Introduction about XRD and there interpretation.
Presented by: Mohumed omar Mahmoud. (Department of pharmaceutics).
RIPER, anantapur.
In this slide contains principle, description of Differential Thermal analysis Application in Polymers.
Presented by: RAMY SALIHEEN (Department of pharmaceutics).
RIPER, anantapur
In this slide contains introduction, principle and applications of differential scanning colorimetry.
Presented by: G.Kavya (Department of pharmaceutics)
RIPER,anantapur.
In this slide contains the deep explanation of Methods of Determination for Drug-Excipient Compatibility Studies.
Presented by: G.Aravind Kumar (Department of industrial pharmacy),
RIPER, anantapur.
In this slide contains principle of IR spectroscopy and sampling techniques.
Presented by: R.Banuteja (Department of pharmaceutical analysis).
RIPER, anantpur.
In this slide contains principle, advantage, dis advantage and application of UPLC.
Presented by: P. Sudheer Kumar. (Department of pharmaceutical analysis)
RIPER, anantapur.
JOURNAL CLUB PRESENTATION (20L81S0402-PA & QA)
Presented by: K VENKATSAI PRASAD (Department of pharmaceutical analysis and quality assurance).RIPER, anantapur
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Slide 1: Title Slide
Extrachromosomal Inheritance
Slide 2: Introduction to Extrachromosomal Inheritance
Definition: Extrachromosomal inheritance refers to the transmission of genetic material that is not found within the nucleus.
Key Components: Involves genes located in mitochondria, chloroplasts, and plasmids.
Slide 3: Mitochondrial Inheritance
Mitochondria: Organelles responsible for energy production.
Mitochondrial DNA (mtDNA): Circular DNA molecule found in mitochondria.
Inheritance Pattern: Maternally inherited, meaning it is passed from mothers to all their offspring.
Diseases: Examples include Leber’s hereditary optic neuropathy (LHON) and mitochondrial myopathy.
Slide 4: Chloroplast Inheritance
Chloroplasts: Organelles responsible for photosynthesis in plants.
Chloroplast DNA (cpDNA): Circular DNA molecule found in chloroplasts.
Inheritance Pattern: Often maternally inherited in most plants, but can vary in some species.
Examples: Variegation in plants, where leaf color patterns are determined by chloroplast DNA.
Slide 5: Plasmid Inheritance
Plasmids: Small, circular DNA molecules found in bacteria and some eukaryotes.
Features: Can carry antibiotic resistance genes and can be transferred between cells through processes like conjugation.
Significance: Important in biotechnology for gene cloning and genetic engineering.
Slide 6: Mechanisms of Extrachromosomal Inheritance
Non-Mendelian Patterns: Do not follow Mendel’s laws of inheritance.
Cytoplasmic Segregation: During cell division, organelles like mitochondria and chloroplasts are randomly distributed to daughter cells.
Heteroplasmy: Presence of more than one type of organellar genome within a cell, leading to variation in expression.
Slide 7: Examples of Extrachromosomal Inheritance
Four O’clock Plant (Mirabilis jalapa): Shows variegated leaves due to different cpDNA in leaf cells.
Petite Mutants in Yeast: Result from mutations in mitochondrial DNA affecting respiration.
Slide 8: Importance of Extrachromosomal Inheritance
Evolution: Provides insight into the evolution of eukaryotic cells.
Medicine: Understanding mitochondrial inheritance helps in diagnosing and treating mitochondrial diseases.
Agriculture: Chloroplast inheritance can be used in plant breeding and genetic modification.
Slide 9: Recent Research and Advances
Gene Editing: Techniques like CRISPR-Cas9 are being used to edit mitochondrial and chloroplast DNA.
Therapies: Development of mitochondrial replacement therapy (MRT) for preventing mitochondrial diseases.
Slide 10: Conclusion
Summary: Extrachromosomal inheritance involves the transmission of genetic material outside the nucleus and plays a crucial role in genetics, medicine, and biotechnology.
Future Directions: Continued research and technological advancements hold promise for new treatments and applications.
Slide 11: Questions and Discussion
Invite Audience: Open the floor for any questions or further discussion on the topic.
Observation of Io’s Resurfacing via Plume Deposition Using Ground-based Adapt...Sérgio Sacani
Since volcanic activity was first discovered on Io from Voyager images in 1979, changes
on Io’s surface have been monitored from both spacecraft and ground-based telescopes.
Here, we present the highest spatial resolution images of Io ever obtained from a groundbased telescope. These images, acquired by the SHARK-VIS instrument on the Large
Binocular Telescope, show evidence of a major resurfacing event on Io’s trailing hemisphere. When compared to the most recent spacecraft images, the SHARK-VIS images
show that a plume deposit from a powerful eruption at Pillan Patera has covered part
of the long-lived Pele plume deposit. Although this type of resurfacing event may be common on Io, few have been detected due to the rarity of spacecraft visits and the previously low spatial resolution available from Earth-based telescopes. The SHARK-VIS instrument ushers in a new era of high resolution imaging of Io’s surface using adaptive
optics at visible wavelengths.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
In silico drugs analogue design: novobiocin analogues.pptx
ZONE ELECTROPHORESIS
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
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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
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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.
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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
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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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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
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
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Although pre-made buffers solutions for electrophoresis are available
in the market.
30
31. RIPER
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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Examples
Tris buffer
Mops buffer
Bis – Tri buffer
ACES buffer
Bicine buffer
CAPS
CAPSO
CHES
Tricine buffer
31
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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
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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.
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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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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 36
Thank You