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ELECTROPHORESIS
Dr. Tintu Jose Manicketh
Assistant Professor
Dept. of Botany
St. Teresa’s College
Ernakulam
Silpa Selvaraj
Roll no: 13
II M.Sc. Botany
St. Teresa’s College
Ernakulam
OVERVIEW
• Agarose gel electrophoresis.
• SDS PAGE electrophoresis.
• Pulse field gel electrophoresis.
2
ELECTROPHORESIS
• Electro means electricity. Phoresis means separation.
• Arne Tiselius(1937) – father of electrophoresis – got Nobel prize in
chemistry for his research on electrophoresis and adsorption analysis,
especially for his discoveries concerning the complex nature of the serum
proteins.
• Electrophoresis is a general term that describes the migration and
separation of charged particles under the influence of an electric field.
• The particles maybe simple ions, complex macromolecules and colloids or
particulate matter- either living cells such as bacteria or inert material such
as oil emulsion, droplet etc.
• The pores present in the gel work like a sieve, allowing the smaller
molecules to pass through more quickly and easily than the larger
molecules.
3
PRINCIPLE
• Electrophoresis relies on the application of an electric field to a gel or a solution.
• The electric field is created by applying a voltage across the electrophoresis apparatus.
• The molecules to be separated must have an electric charge.
• For example, DNA and RNA are negatively charged due to their phosphate groups, while
proteins can be positively or negatively charged depending on their amino acid
composition.
• When the electric field is applied, the charged molecules move through the gel or
solution.
• Negatively charged molecules (anions) migrate towards the positive electrode (anode),
while positively charged molecules (cations) move towards the negative electrode
(cathode).
4
• The molecules move at different speeds through the gel or solution, primarily
based on their size and charge.
• Smaller molecules move more quickly, while larger ones move more slowly.
• This differential migration leads to the separation of molecules along the gel.
• After electrophoresis, the separated molecules can be visualized by various
techniques, such as staining with specific dyes or using techniques like
autoradiography for radioactive molecules.
5
The rate of migration of a particle in the electrical field depend on factors;
• Net charge of the molecule.
• Size and shape of particle.
• Properties of supporting medium.
• Temperature of operation.
6
GEL ELECTROPHORESIS
• Gel electrophoresis is a simple, rapid and sensitive analytical technique for the
separation of charged particle.
• The gels are porous and the size of the pores relative to that of the molecule
determines whether the molecule will enter the pore and be retarded or will bypass
it.
• The separation thus not only depends on the charge on the molecule but also on its
size.
APPARATUS OF GEL ELECTROPHORESIS
• Vertical gel apparatus: It is commonly used in SDS PAGE for the separation of
proteins.
• Horizontal gel apparatus: It is used for immuno electrophoresis and
electrophoresis of DNA and RNA in the agarose gel.
7
Vertical system Horizontal system
8
Agarose gel electrophoresis
9
• Agarose gel is the supporting media in
agarose gel electrophoresis.
• For the electrophoresis
of DNA, RNA and Protein agarose
gel is used.
AGAROSE
• Agarose is polysaccharide which is isolated from seaweeds.
• It is a natural polymer made up of alternating Beta-D-galactose
and 3,6-anhydro-L-galactose units od agarobiose.
• This gel has generally larger pore size, which makes them
suitable to separate larger molecules having molecular mass
more than 200 kDa.
• It is most commonly used for the electrophoresis of both protein
and nucleic acids.
• Agarose is used in concentration between 1% and 3%.
10
PRINCIPLE
• Sample is pipetted into the sample wells, followed by the application of an electric
current which causes the negatively-charged DNA to migrate towards the anode.
• The rate of migration is proportional to size, shape, mass etc.
• Larger molecules have difficulty in moving through the pore size of the supporting
media, whereas the smaller molecules has more mobility through it.
• The bands of protein or nucleic acid is visualized by using intercalating dye, i.e.,
ethidium bromide (Etbr), they are visualized by fluorescence when illuminated with
ultraviolet light.
• DNA fragments take up the dye as they migrate through the gel.
• The larger fragments of DNA fluoresce more intensely. The smaller fragments
fluoresce less intensely.
• A ladder set of DNA of known size can be run simultaneously and used to estimate
the sizes of the other unknown fragments.
11
REQUIREMENTS
12
• An electrophoresis chamber and power supply.
• Gel casting trays, which are available in a variety of sizes and composed of UV
transparent plastic.
• The open ends of the trays are closed with tape while the gel is being cast, then
removed prior to electrophoresis.
• Sample combs, around which molten medium is poured to form sample wells in the
gel.
• Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA
(TBE).
• Loading buffer, contains something dense (e.g. glycerol) to allow the sample
to fall into the sample wells, and one or two tracking dyes, which migrate in to
the gel and allow visual monitoring. Usually consists of:-
• Salts: It creates environment with favorable ionic strength and pH. e.g., Tris-
HCl.
• Metal chelator: It prevents nucleases that degrade the nucleic acid such as
EDTA.
• Staining: DNA molecules are easily visualized under an ultraviolet lamp when
electrphoresed in the presence of the extrinsic fluor ethidium bromide.
Alternatively, nucleic acids can be stained after electrophoretic separation by
soaking the gel in a solution of ethidium bromide. When intercalated into
doublestranded DNA, fluorescence of this molecule increases greatly. It is also
possible to detect DNA with the extrinsic fluor 1-anilino 8-naphthalene
sulphonate.
• Transilluminator is used to visualize stained DNA in gels.
13
ELECTROPHORETIC APPARATUS
14
UV Transilluminator
• An ultra-violet (UV) transilluminator is an equipment used
in life science laboratories for visualization of target DNAs
and proteins.
• Sometimes referred to as a gel light box or lab light box.
• It works by emitting high levels of UV radiation through
the viewing surface.
• The key application for a UV transilluminator is for
visualization of DNA and protein in agarose and
polyacrylamide gels after electrophoresis.
• Gels can be directly placed onto the UV transilluminator;
wavelength will vary on your particular application.
15
PROCEDURE
1. To prepare gel, agarose powder is mixed with electrophoresis buffer to the desired
concentration, and heated in a microwave oven to melt it.
• The lower the concentration of agarose, the faster the DNA fragments migrate.
• If the aim is to separate large DNA fragments, a low concentration of agarose
should be used, and if the aim is to separate small DNA fragments, a high
concentration of agarose is recommended.
2. Ethidium bromide is added to the gel to facilitate visualization of DNA after
electrophoresis.
3. After cooling the solution to about 60oC, it is poured into a casting tray
containing a sample comb and allowed to solidify at room temperature.
4. After the gel has solidified, the comb is removed, taking care not to rip the bottom
of the wells.
16
• Samples containing DNA mixed with loading buffer are then pipetted into the
sample wells, the lid and power leads are placed on the apparatus, and a current is
applied.
• The current flow can be confirmed by observing bubbles coming off the
electrodes.
• DNA will migrate towards the positive electrode.
• The distance DNA has migrated in the gel can be judged by visually monitoring
migration of the tracking dyes like bromophenol blue and xylene cyanol dyes.
17
APPLICATIONS
• Agarose gel electrophoresis is a routinely used method for separating proteins, DNA
or RNA.
• Estimation of the size of DNA molecules.
• Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic
fingerprinting
• Separation of restricted genomic DNA prior to Southern analysis, or of RNA prior to
Northern analysis.
• Employed to estimate the size of DNA fragments after digesting with restriction
enzymes, e.g. in restriction mapping of cloned DNA.
• Used to resolve circular DNA with different supercoiling topology, and to resolve
fragments that differ due to DNA synthesis.
18
Advantages
• For most applications, only a single-component agarose is needed and no
polymerization catalysts are required.
• Agarose gels are simple and rapid to prepare.
• The gel is easily poured, does not denature the samples.
• The samples can also be recovered.
Disadvantages
• Gels can melt during electrophoresis.
• The buffer can become exhausted.
• Different forms of genetic material may run in unpredictable forms.
19
SDS PAGE ELECTROPHORESIS
20
• SDS – PAGE or sodium dodecyl sulfate-polyacrylamide gel
electrophoresis is a technique most commonly used in
genetics, biotechnology, biochemistry, and molecular
biology laboratories for the separation of proteins from a
mixed sample.
• They separate proteins on the basis of their electrophoretic
mobility in which the mobility of the molecule is inversely
proportional to its size and directly proportional to its
charge.
• It involves the use of vertical gel apparatus to separate
proteins.
POLYACRYLAMIDE GEL
• Polyacrylamide gel consist of chains of acrylamide monomers crosslinked
with N, N’-methylenebisacrylamide units, which is commonly termed as
bisacrylamide.
• The degree of cross-linking determines the hardness and softness of the gel
which can be made by adjusting its concentration.
• The more the cross-linking, the harder will be the gel which slows down the
migration of molecules but with the loose gel, the molecules migrate faster.
• In this gel, pore size and resolving power totally depends upon the
concentration of acrylamide and bisacrylamide.
• The concentration of the gel normally varies from 5% to 25%.
21
PRINCIPLE
• The main principle of SDS – PAGE is to separate specific proteins
electrophoretically from a mixture of samples according to their size.
• Polyacrylamide is a gel-like matrix suitable for the separation of proteins which is
a product of polymerization reaction between acrylamide and N, N’ –methylene-
bis-acrylamide (BIS).
• The degree of cross-linking determines the hardness and softness of the gel which
can be made by adjusting its concentration.
• The more the cross-linking, the harder will be the gel which slows down the
migration of molecules but with the loose gel, the molecules migrate faster.
• SDS is an anionic detergent which binds to protein backbone and forms a
negative charge which breaks the bonds of proteins disrupting the protein
structure.
• Then the protein gets separated electrophoretically based on the length of their
polypeptide chain.
22
REQUIREMENTS
• Gel or media acrylamide solutions.
• Buffer system the separation and migration patterns of proteins in gel
electrophoresis are determined by the chemical composition and pH of the buffer
system.
• Staining dye
• Protein samples
• Molecular weight markers
• An electrophoresis chamber and power supply
• Glass plates (a short and a top plate)
• Casting frame
• Casting stand
• Combs
23
PROCEDURE
Sample preparation
• Samples may be any material containing proteins or nucleic acids.
• The sample to analyze is optionally mixed with a chemical denaturant,
usually SDS for proteins or urea for nucleic acids.
• SDS is an anionic detergent that denatures secondary structures, and additionally
applies a negative charge to each protein in proportion to its mass.
• Urea breaks the hydrogen bonds between the base pairs of the nucleic acid,
causing the constituent strands to anneal.
• With the protein sample, the buffer solution is added in microcentrifuge tubes and
heat at 100°C for 3 minutes.
• Then the tubes are centrifuged at 15,000 rpm for 1 minute at 4°C.
• The supernatant is used further in SDS – PAGE processes.
24
Gel preparation
• For preparing an electrophoretic gel, several components including acrylamide, N,
N’ –methylene-bis-acrylamide (BIS), and a buffer solution is mixed together.
• During polymerization of the gel, ammonium persulfate, a free radical source, and
a stabilizer is added and the mixture is degassed or butanol is added to prevent
bubble formation.
• A comb is inserted between the spaces of the glass plate and allowed to
polymerize.
• The polymerized gel is called a gel cassette.
• After the gel is polymerized the comb can be removed and the gel is ready for
electrophoresis.
25
Electrophoresis
• The denatured 30 ml sample is pipetted out and placed in the well and
allowed to run at 30 mA for about one hour.
• As an electric current is applied, negatively charged protein molecules
migrate towards a positively charged electrode through the gel.
• Each protein molecules also migrate at a different rate based on its
molecular size.
• Smaller biomolecules travel farther down the gel, while larger ones remain
closer to the point of origin.
• High voltage also increases the rate of migration.
• For protein molecules to be completely separated, it may take about one
hour which is then stained and visualized.
26
Detection
• Following electrophoresis, the gel may be stained, allowing visualization of the
separated proteins.
• After staining, different species biomolecules appear as distinct bands within the
gel.
• It is common to run molecular weight size markers of known molecular weight in
a separate lane in the gel to calibrate the gel and determine the
approximate molecular mass of unknown biomolecules by comparing the distance
traveled relative to the marker.
27
APPLICATIONS
• Measuring molecular weight.
• Peptide mapping.
• Estimation of protein size.
• Determination of protein subunits or aggregation structures.
• Estimation of protein purity.
• Protein quantification.
• Monitoring protein integrity.
• Comparison of the polypeptide composition of different samples.
• Analysis of the number and size of polypeptide subunits.
• Post-electrophoresis applications, such as Western blotting.
• Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes.
• Detection of Protein Ubiquitination.
28
Advantages:
• SDS – PAGE is a highly sensitive test that separate molecules that have a minimum (~2%)
difference in mass.
• Even small amount of sample is enough for processing.
• Pure DNA can be recovered from the gel.
• The pore size of the polyacrylamide gel can be controlled by adjusting the concentration of
the monomers.
Disadvantages:
• Gels are often difficult to prepare and takes a long time.
• Monomers used are potent neurotoxin chemical.
• Resolution of band is poor due to high alkaline operating pH.
• New gel is needed for each experiment.
29
PULSE FIELD GEL ELECTROPHORESIS
• Pulse – field gel electrophoresis is a separation
technique of large DNA molecules.
• The DNA molecules are digested with unique
restriction enzymes and are separated under the
electric field that changes direction periodically.
30
PRINCIPLE
• In PFGE, an electric field is applied to the gel in a pulsed manner. This means that
the direction of the electric field is altered at regular intervals. This prevents large
DNA fragments from migrating too quickly through the gel and allows them to
reorient themselves, improving separation.
• The DNA samples are embedded in an agarose gel. Agarose gels are chosen because
they have larger pores, allowing large DNA fragments to move more freely.
• In pulsed field gel electrophoresis, large DNA molecules with above 30 to 50 kb
length fragment runs on the gel matrix at the same rate and appears as a single large
diffuse band.
• The various length of the DNA changes with the periodically changing direction of
electric field where large DNA molecule moves slowly when the direction of field is
changed while small DNA molecule moves faster.
• This separation of molecule continues over the course of time with the consistent
changing of direction.
• The DNA fragments are visualized using DNA staining methods, usually ethidium
bromide. The result is a distinct banding pattern, with larger fragments closer to the
origin and smaller fragments farther from the origin.
31
Why pulse field gel electrophoresis over standard gel
electrophoresis technique?
32
• The standard gel electrophoresis only separates small molecules of DNA and is
unable to separate DNA molecules larger than 15 to 20kb effectively.
• In 1984, David C. Schwartz and Charles Cantor developed an electrophoresis
technique that can separate the DNA molecules larger than 50kb which became
known as pulsed field gel electrophoresis.
• The development of PFGE provided a huge advantage for molecular biology
research and expanded the range of resolution for DNA fragments.
PROCEDURE
• The concept of standard gel agarose electrophoresis and pulsed field gel
electrophoresis is similar but equipment required to run PFGE is much more
complicated and needs a trained personnel.
• Instead of constantly running the voltage in one direction, the voltage is
periodically switched among three directions; one that runs through the central
axis of the gel and two that run at an angle of 60 degrees either side.
• The pulse times are equal for each direction resulting in a net forward migration of
the DNA.
33
Lysis
• Bacterial cells are taken from an agar plate and are mixed and loaded with melted
agarose.
• Bacterial cells are immobilized into agarose blocks known as plug mold to protect
the chromosomal DNA from mechanical damage.
• This is the first step where bacterial cells are lysed and DNA is released in the
agarose plug.
34
Digestion of DNA
• The bacterial DNA is treated with unusual cutting restriction enzymes so that it
yields less number of larger size DNA fragments.
Electrophoresis
• The larger pieces of DNA are subjected to pulse field gel electrophoresis by applying
electric current and altering its direction at regular intervals where the DNA
fragments are separated on the basis of their size. (in contrast to the conventional
agarose gel electrophoresis done to separate the smaller fragments where the current
is applied in a single direction).
35
Visualization
• The gel is stained with a SYBR Green I where the DNA are visualized under the
ultraviolet (UV) light.
• The bands of the fragment of DNA molecules of different organisms are analyzed and
compared to standards manually.
• Computer software like BioNumerics is also used.
• A digital camera is also used to take a photograph of the gel.
36
APPLICATIONS
• Since, field gel electrophoresis allows the separation of DNA fragments containing up
to 100,000 bp (100 kilobase pairs, or kbp), characterization of such large fragments has
allowed construction of a physical map for the chromosomes from several bacterial
species.
• It is commonly considered a gold standard in epidemiological studies of pathogenic
organisms.
• In genome size estimation, PFGE is considered as an efficient method.
• PFGE is also used for genotyping or genetic fingerprinting.
• Yeast Artificial Chromosome (YAC) libraries are constructed by PFGE.
• PFGE has also been used in the analysis of large DNA molecules from fungi and
parasitic protozoa.
• PFGE is also used in the study of radiation-induced DNA damage and repair.
37
Advantages
• PFGE hold the strength of separating large DNA molecules to over 10 Mb pairs.
• PFGE is used in the sub typing of many pathogenic bacteria and are mainly used
in epidemiological field.
• PFGE follows the similar basic format as a universal generic method for bacterial
sub typing and only requires a choice of restriction enzymes and condition of
electrophoresis for each sample species.
• The restriction patterns generated by PFGE are stable and can be reproduced.
38
Disadvantages
• PFGE method consumes more time which might take overnight or a couple of
days.
• It cannot separate the fragments in every part of the gel at the same time.
• One change in restriction site might form more than one band.
• Not all strains can be sub typed by PFGE.
• PFGE does not differentiate isolates to the same degree as whole genome
sequencing (WGE).
• Requires trained and skilled technicians.
• Pattern results vary slightly between technicians.
• Bands are not independent.
• Change in one restriction site can mean more than one band change.
39
REFERENCE
• Aryal, S. (2022, January). Agarose Gel Electrophoresis-
Definition,Principle,Parts,Steps and Applications. Retrieved from Microbe Notes:
https://microbenotes.com/agarose-gel-electrophoresis/
• Binod, G.C. (2021, February). SDS PAGE: Introduction,Principle,Methodology
and Applications. Retrieved from The Science Notes: https: // thesciencenotes.com
/sds-page
• Dura, S. (2022, January). Pulse Field Gel Electrophoresis (PFGE). Retrieved from
The Science Notes: http://www.thesciencenotes.com
• https://www.biotechnologynotes.com/electrophoresis/electrophoresis-meaning-
definition-and-classification-withdiagram/293
• https://www.researchgate.net/publication/310994699_Fundamentals_and_Techniq
ues_of_Biophysics_and_Molecular_Biology
40
Thank you
41

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electrophoresis: types, advantages, disadvantages and applications.

  • 1. ELECTROPHORESIS Dr. Tintu Jose Manicketh Assistant Professor Dept. of Botany St. Teresa’s College Ernakulam Silpa Selvaraj Roll no: 13 II M.Sc. Botany St. Teresa’s College Ernakulam
  • 2. OVERVIEW • Agarose gel electrophoresis. • SDS PAGE electrophoresis. • Pulse field gel electrophoresis. 2
  • 3. ELECTROPHORESIS • Electro means electricity. Phoresis means separation. • Arne Tiselius(1937) – father of electrophoresis – got Nobel prize in chemistry for his research on electrophoresis and adsorption analysis, especially for his discoveries concerning the complex nature of the serum proteins. • Electrophoresis is a general term that describes the migration and separation of charged particles under the influence of an electric field. • The particles maybe simple ions, complex macromolecules and colloids or particulate matter- either living cells such as bacteria or inert material such as oil emulsion, droplet etc. • The pores present in the gel work like a sieve, allowing the smaller molecules to pass through more quickly and easily than the larger molecules. 3
  • 4. PRINCIPLE • Electrophoresis relies on the application of an electric field to a gel or a solution. • The electric field is created by applying a voltage across the electrophoresis apparatus. • The molecules to be separated must have an electric charge. • For example, DNA and RNA are negatively charged due to their phosphate groups, while proteins can be positively or negatively charged depending on their amino acid composition. • When the electric field is applied, the charged molecules move through the gel or solution. • Negatively charged molecules (anions) migrate towards the positive electrode (anode), while positively charged molecules (cations) move towards the negative electrode (cathode). 4
  • 5. • The molecules move at different speeds through the gel or solution, primarily based on their size and charge. • Smaller molecules move more quickly, while larger ones move more slowly. • This differential migration leads to the separation of molecules along the gel. • After electrophoresis, the separated molecules can be visualized by various techniques, such as staining with specific dyes or using techniques like autoradiography for radioactive molecules. 5
  • 6. The rate of migration of a particle in the electrical field depend on factors; • Net charge of the molecule. • Size and shape of particle. • Properties of supporting medium. • Temperature of operation. 6
  • 7. GEL ELECTROPHORESIS • Gel electrophoresis is a simple, rapid and sensitive analytical technique for the separation of charged particle. • The gels are porous and the size of the pores relative to that of the molecule determines whether the molecule will enter the pore and be retarded or will bypass it. • The separation thus not only depends on the charge on the molecule but also on its size. APPARATUS OF GEL ELECTROPHORESIS • Vertical gel apparatus: It is commonly used in SDS PAGE for the separation of proteins. • Horizontal gel apparatus: It is used for immuno electrophoresis and electrophoresis of DNA and RNA in the agarose gel. 7
  • 9. Agarose gel electrophoresis 9 • Agarose gel is the supporting media in agarose gel electrophoresis. • For the electrophoresis of DNA, RNA and Protein agarose gel is used.
  • 10. AGAROSE • Agarose is polysaccharide which is isolated from seaweeds. • It is a natural polymer made up of alternating Beta-D-galactose and 3,6-anhydro-L-galactose units od agarobiose. • This gel has generally larger pore size, which makes them suitable to separate larger molecules having molecular mass more than 200 kDa. • It is most commonly used for the electrophoresis of both protein and nucleic acids. • Agarose is used in concentration between 1% and 3%. 10
  • 11. PRINCIPLE • Sample is pipetted into the sample wells, followed by the application of an electric current which causes the negatively-charged DNA to migrate towards the anode. • The rate of migration is proportional to size, shape, mass etc. • Larger molecules have difficulty in moving through the pore size of the supporting media, whereas the smaller molecules has more mobility through it. • The bands of protein or nucleic acid is visualized by using intercalating dye, i.e., ethidium bromide (Etbr), they are visualized by fluorescence when illuminated with ultraviolet light. • DNA fragments take up the dye as they migrate through the gel. • The larger fragments of DNA fluoresce more intensely. The smaller fragments fluoresce less intensely. • A ladder set of DNA of known size can be run simultaneously and used to estimate the sizes of the other unknown fragments. 11
  • 12. REQUIREMENTS 12 • An electrophoresis chamber and power supply. • Gel casting trays, which are available in a variety of sizes and composed of UV transparent plastic. • The open ends of the trays are closed with tape while the gel is being cast, then removed prior to electrophoresis. • Sample combs, around which molten medium is poured to form sample wells in the gel. • Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE).
  • 13. • Loading buffer, contains something dense (e.g. glycerol) to allow the sample to fall into the sample wells, and one or two tracking dyes, which migrate in to the gel and allow visual monitoring. Usually consists of:- • Salts: It creates environment with favorable ionic strength and pH. e.g., Tris- HCl. • Metal chelator: It prevents nucleases that degrade the nucleic acid such as EDTA. • Staining: DNA molecules are easily visualized under an ultraviolet lamp when electrphoresed in the presence of the extrinsic fluor ethidium bromide. Alternatively, nucleic acids can be stained after electrophoretic separation by soaking the gel in a solution of ethidium bromide. When intercalated into doublestranded DNA, fluorescence of this molecule increases greatly. It is also possible to detect DNA with the extrinsic fluor 1-anilino 8-naphthalene sulphonate. • Transilluminator is used to visualize stained DNA in gels. 13
  • 15. UV Transilluminator • An ultra-violet (UV) transilluminator is an equipment used in life science laboratories for visualization of target DNAs and proteins. • Sometimes referred to as a gel light box or lab light box. • It works by emitting high levels of UV radiation through the viewing surface. • The key application for a UV transilluminator is for visualization of DNA and protein in agarose and polyacrylamide gels after electrophoresis. • Gels can be directly placed onto the UV transilluminator; wavelength will vary on your particular application. 15
  • 16. PROCEDURE 1. To prepare gel, agarose powder is mixed with electrophoresis buffer to the desired concentration, and heated in a microwave oven to melt it. • The lower the concentration of agarose, the faster the DNA fragments migrate. • If the aim is to separate large DNA fragments, a low concentration of agarose should be used, and if the aim is to separate small DNA fragments, a high concentration of agarose is recommended. 2. Ethidium bromide is added to the gel to facilitate visualization of DNA after electrophoresis. 3. After cooling the solution to about 60oC, it is poured into a casting tray containing a sample comb and allowed to solidify at room temperature. 4. After the gel has solidified, the comb is removed, taking care not to rip the bottom of the wells. 16
  • 17. • Samples containing DNA mixed with loading buffer are then pipetted into the sample wells, the lid and power leads are placed on the apparatus, and a current is applied. • The current flow can be confirmed by observing bubbles coming off the electrodes. • DNA will migrate towards the positive electrode. • The distance DNA has migrated in the gel can be judged by visually monitoring migration of the tracking dyes like bromophenol blue and xylene cyanol dyes. 17
  • 18. APPLICATIONS • Agarose gel electrophoresis is a routinely used method for separating proteins, DNA or RNA. • Estimation of the size of DNA molecules. • Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting • Separation of restricted genomic DNA prior to Southern analysis, or of RNA prior to Northern analysis. • Employed to estimate the size of DNA fragments after digesting with restriction enzymes, e.g. in restriction mapping of cloned DNA. • Used to resolve circular DNA with different supercoiling topology, and to resolve fragments that differ due to DNA synthesis. 18
  • 19. Advantages • For most applications, only a single-component agarose is needed and no polymerization catalysts are required. • Agarose gels are simple and rapid to prepare. • The gel is easily poured, does not denature the samples. • The samples can also be recovered. Disadvantages • Gels can melt during electrophoresis. • The buffer can become exhausted. • Different forms of genetic material may run in unpredictable forms. 19
  • 20. SDS PAGE ELECTROPHORESIS 20 • SDS – PAGE or sodium dodecyl sulfate-polyacrylamide gel electrophoresis is a technique most commonly used in genetics, biotechnology, biochemistry, and molecular biology laboratories for the separation of proteins from a mixed sample. • They separate proteins on the basis of their electrophoretic mobility in which the mobility of the molecule is inversely proportional to its size and directly proportional to its charge. • It involves the use of vertical gel apparatus to separate proteins.
  • 21. POLYACRYLAMIDE GEL • Polyacrylamide gel consist of chains of acrylamide monomers crosslinked with N, N’-methylenebisacrylamide units, which is commonly termed as bisacrylamide. • The degree of cross-linking determines the hardness and softness of the gel which can be made by adjusting its concentration. • The more the cross-linking, the harder will be the gel which slows down the migration of molecules but with the loose gel, the molecules migrate faster. • In this gel, pore size and resolving power totally depends upon the concentration of acrylamide and bisacrylamide. • The concentration of the gel normally varies from 5% to 25%. 21
  • 22. PRINCIPLE • The main principle of SDS – PAGE is to separate specific proteins electrophoretically from a mixture of samples according to their size. • Polyacrylamide is a gel-like matrix suitable for the separation of proteins which is a product of polymerization reaction between acrylamide and N, N’ –methylene- bis-acrylamide (BIS). • The degree of cross-linking determines the hardness and softness of the gel which can be made by adjusting its concentration. • The more the cross-linking, the harder will be the gel which slows down the migration of molecules but with the loose gel, the molecules migrate faster. • SDS is an anionic detergent which binds to protein backbone and forms a negative charge which breaks the bonds of proteins disrupting the protein structure. • Then the protein gets separated electrophoretically based on the length of their polypeptide chain. 22
  • 23. REQUIREMENTS • Gel or media acrylamide solutions. • Buffer system the separation and migration patterns of proteins in gel electrophoresis are determined by the chemical composition and pH of the buffer system. • Staining dye • Protein samples • Molecular weight markers • An electrophoresis chamber and power supply • Glass plates (a short and a top plate) • Casting frame • Casting stand • Combs 23
  • 24. PROCEDURE Sample preparation • Samples may be any material containing proteins or nucleic acids. • The sample to analyze is optionally mixed with a chemical denaturant, usually SDS for proteins or urea for nucleic acids. • SDS is an anionic detergent that denatures secondary structures, and additionally applies a negative charge to each protein in proportion to its mass. • Urea breaks the hydrogen bonds between the base pairs of the nucleic acid, causing the constituent strands to anneal. • With the protein sample, the buffer solution is added in microcentrifuge tubes and heat at 100°C for 3 minutes. • Then the tubes are centrifuged at 15,000 rpm for 1 minute at 4°C. • The supernatant is used further in SDS – PAGE processes. 24
  • 25. Gel preparation • For preparing an electrophoretic gel, several components including acrylamide, N, N’ –methylene-bis-acrylamide (BIS), and a buffer solution is mixed together. • During polymerization of the gel, ammonium persulfate, a free radical source, and a stabilizer is added and the mixture is degassed or butanol is added to prevent bubble formation. • A comb is inserted between the spaces of the glass plate and allowed to polymerize. • The polymerized gel is called a gel cassette. • After the gel is polymerized the comb can be removed and the gel is ready for electrophoresis. 25
  • 26. Electrophoresis • The denatured 30 ml sample is pipetted out and placed in the well and allowed to run at 30 mA for about one hour. • As an electric current is applied, negatively charged protein molecules migrate towards a positively charged electrode through the gel. • Each protein molecules also migrate at a different rate based on its molecular size. • Smaller biomolecules travel farther down the gel, while larger ones remain closer to the point of origin. • High voltage also increases the rate of migration. • For protein molecules to be completely separated, it may take about one hour which is then stained and visualized. 26
  • 27. Detection • Following electrophoresis, the gel may be stained, allowing visualization of the separated proteins. • After staining, different species biomolecules appear as distinct bands within the gel. • It is common to run molecular weight size markers of known molecular weight in a separate lane in the gel to calibrate the gel and determine the approximate molecular mass of unknown biomolecules by comparing the distance traveled relative to the marker. 27
  • 28. APPLICATIONS • Measuring molecular weight. • Peptide mapping. • Estimation of protein size. • Determination of protein subunits or aggregation structures. • Estimation of protein purity. • Protein quantification. • Monitoring protein integrity. • Comparison of the polypeptide composition of different samples. • Analysis of the number and size of polypeptide subunits. • Post-electrophoresis applications, such as Western blotting. • Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes. • Detection of Protein Ubiquitination. 28
  • 29. Advantages: • SDS – PAGE is a highly sensitive test that separate molecules that have a minimum (~2%) difference in mass. • Even small amount of sample is enough for processing. • Pure DNA can be recovered from the gel. • The pore size of the polyacrylamide gel can be controlled by adjusting the concentration of the monomers. Disadvantages: • Gels are often difficult to prepare and takes a long time. • Monomers used are potent neurotoxin chemical. • Resolution of band is poor due to high alkaline operating pH. • New gel is needed for each experiment. 29
  • 30. PULSE FIELD GEL ELECTROPHORESIS • Pulse – field gel electrophoresis is a separation technique of large DNA molecules. • The DNA molecules are digested with unique restriction enzymes and are separated under the electric field that changes direction periodically. 30
  • 31. PRINCIPLE • In PFGE, an electric field is applied to the gel in a pulsed manner. This means that the direction of the electric field is altered at regular intervals. This prevents large DNA fragments from migrating too quickly through the gel and allows them to reorient themselves, improving separation. • The DNA samples are embedded in an agarose gel. Agarose gels are chosen because they have larger pores, allowing large DNA fragments to move more freely. • In pulsed field gel electrophoresis, large DNA molecules with above 30 to 50 kb length fragment runs on the gel matrix at the same rate and appears as a single large diffuse band. • The various length of the DNA changes with the periodically changing direction of electric field where large DNA molecule moves slowly when the direction of field is changed while small DNA molecule moves faster. • This separation of molecule continues over the course of time with the consistent changing of direction. • The DNA fragments are visualized using DNA staining methods, usually ethidium bromide. The result is a distinct banding pattern, with larger fragments closer to the origin and smaller fragments farther from the origin. 31
  • 32. Why pulse field gel electrophoresis over standard gel electrophoresis technique? 32 • The standard gel electrophoresis only separates small molecules of DNA and is unable to separate DNA molecules larger than 15 to 20kb effectively. • In 1984, David C. Schwartz and Charles Cantor developed an electrophoresis technique that can separate the DNA molecules larger than 50kb which became known as pulsed field gel electrophoresis. • The development of PFGE provided a huge advantage for molecular biology research and expanded the range of resolution for DNA fragments.
  • 33. PROCEDURE • The concept of standard gel agarose electrophoresis and pulsed field gel electrophoresis is similar but equipment required to run PFGE is much more complicated and needs a trained personnel. • Instead of constantly running the voltage in one direction, the voltage is periodically switched among three directions; one that runs through the central axis of the gel and two that run at an angle of 60 degrees either side. • The pulse times are equal for each direction resulting in a net forward migration of the DNA. 33
  • 34. Lysis • Bacterial cells are taken from an agar plate and are mixed and loaded with melted agarose. • Bacterial cells are immobilized into agarose blocks known as plug mold to protect the chromosomal DNA from mechanical damage. • This is the first step where bacterial cells are lysed and DNA is released in the agarose plug. 34
  • 35. Digestion of DNA • The bacterial DNA is treated with unusual cutting restriction enzymes so that it yields less number of larger size DNA fragments. Electrophoresis • The larger pieces of DNA are subjected to pulse field gel electrophoresis by applying electric current and altering its direction at regular intervals where the DNA fragments are separated on the basis of their size. (in contrast to the conventional agarose gel electrophoresis done to separate the smaller fragments where the current is applied in a single direction). 35
  • 36. Visualization • The gel is stained with a SYBR Green I where the DNA are visualized under the ultraviolet (UV) light. • The bands of the fragment of DNA molecules of different organisms are analyzed and compared to standards manually. • Computer software like BioNumerics is also used. • A digital camera is also used to take a photograph of the gel. 36
  • 37. APPLICATIONS • Since, field gel electrophoresis allows the separation of DNA fragments containing up to 100,000 bp (100 kilobase pairs, or kbp), characterization of such large fragments has allowed construction of a physical map for the chromosomes from several bacterial species. • It is commonly considered a gold standard in epidemiological studies of pathogenic organisms. • In genome size estimation, PFGE is considered as an efficient method. • PFGE is also used for genotyping or genetic fingerprinting. • Yeast Artificial Chromosome (YAC) libraries are constructed by PFGE. • PFGE has also been used in the analysis of large DNA molecules from fungi and parasitic protozoa. • PFGE is also used in the study of radiation-induced DNA damage and repair. 37
  • 38. Advantages • PFGE hold the strength of separating large DNA molecules to over 10 Mb pairs. • PFGE is used in the sub typing of many pathogenic bacteria and are mainly used in epidemiological field. • PFGE follows the similar basic format as a universal generic method for bacterial sub typing and only requires a choice of restriction enzymes and condition of electrophoresis for each sample species. • The restriction patterns generated by PFGE are stable and can be reproduced. 38
  • 39. Disadvantages • PFGE method consumes more time which might take overnight or a couple of days. • It cannot separate the fragments in every part of the gel at the same time. • One change in restriction site might form more than one band. • Not all strains can be sub typed by PFGE. • PFGE does not differentiate isolates to the same degree as whole genome sequencing (WGE). • Requires trained and skilled technicians. • Pattern results vary slightly between technicians. • Bands are not independent. • Change in one restriction site can mean more than one band change. 39
  • 40. REFERENCE • Aryal, S. (2022, January). Agarose Gel Electrophoresis- Definition,Principle,Parts,Steps and Applications. Retrieved from Microbe Notes: https://microbenotes.com/agarose-gel-electrophoresis/ • Binod, G.C. (2021, February). SDS PAGE: Introduction,Principle,Methodology and Applications. Retrieved from The Science Notes: https: // thesciencenotes.com /sds-page • Dura, S. (2022, January). Pulse Field Gel Electrophoresis (PFGE). Retrieved from The Science Notes: http://www.thesciencenotes.com • https://www.biotechnologynotes.com/electrophoresis/electrophoresis-meaning- definition-and-classification-withdiagram/293 • https://www.researchgate.net/publication/310994699_Fundamentals_and_Techniq ues_of_Biophysics_and_Molecular_Biology 40