Electrophoresis is a laboratory technique used to separate DNA, RNA, or protein molecules based on their size and electrical charge. Different types of electrophoresis. Gel electrophoresis; Agarose Gel electrophoresis; polyacrylamide gel electrophoresis; pulsed-field gel electrophoresis

1. ELECTROPHORESIS
Tasmina Ferdous Susmi
Dept. of Genetic Engineering and Biotechnology
Jashore University of Science and Technology, Bangladesh

2. Index
 Electrophoresis
 Gel Electrophoresis
 Agarose Gel Electrophoresis
 Polyacrylamide Gel Electrophoresis
 Pulsed Field Gel Electrophoresis

3. Electrophoresis
 A laboratory technique used to separate DNA, RNA, or protein molecules based on their size
and electrical charge.
 Term electrophoresis describes the migration of charged particle under the influence of an
electric field
 An electric current is used to move molecules to be separated through a medium.
 Pores in the gel work like a sieve, allowing smaller molecules to move faster than larger
molecules.
 The conditions used during electrophoresis can be adjusted to separate molecules in a
desired size range.

4. Principle of Electrophoresis
 The separation or purification technique of proteins, DNA, and RNA that differ in charge, size,
and conformation.
 Charged molecules are placed at one end according to their charge and an electric field is
applied.
 Movement of charged species in an electric field gives differential mobility to the sample
molecule based on the charge and consequently resolve them
 On passing current, they start migrating towards opposite electrode which can be either
positive or negative electrode.
 The size, shape, and charge of the molecule remain constant during electrophoresis and
determines the mobility of ionic particles.

5. Principle of Electrophoresis

6. Factors affecting Electrophoresis
Electric Field
• Voltage
• Current
• Resistance
Sample
• Charge
• Size
• Shape
Buffer
• Composition
• Concentratio
n
• pH
Supporting
Medium
• Medium Type
• Electro
osmosis

7. Types of Electrophoresis
Electrophoresis
Slab
Electrophoresis
Zone
Electrophoresis
Immuno
electrophoresis
Isoelectro
focusing
Capillary
Electrophoresis
Gel
Electrophoresis
Agarose Gel
Electrophoresis
Polyacrylamide Gel
Electrophoresis
Pulse Field Gel
Electrophoresis
Paper
Electrophoresis

8. Gel Electrophoresis
 A simple, rapid and sensitive analytical technique for the separation of charged particle.
 The gels, however, 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.
 Needless to say, that resolution of a sample is sharper and better in a gel than in any other
type of medium.
 Samples are loaded into wells (indentations) at one end of a gel, and an electric current is
applied to pull them through the gel.
 There is difference in the electrophoretic mobility of these charged molecules due to their
difference in size, shape, and charge.

9. Gel Electrophoresis: Two Types of Materials Are Used to Make
Gels
 Agarose
 Agarose is natural colloid which is isolated from the seaweed.
 It is linear polysaccharide.
 It is made up of repeating units of agarobiose, comprises alternating units of 3,6-anhydrolactose
and galactose.
 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. Gel Electrophoresis: Two Types of Materials Are Used to Make
Gels
 Polyacrylamide
 Polyacrylamide gel is consisting of chains of acrylamide monomers crosslinked with N, N’-
methylenebisacrylamide units, which is commonly termed as bisacrylamide.
 In this gel, pore size and resolving power is totally depends upon the concentration of
acrylamide and bisacrylamide.
 The concentration of the gel normally varies from 5% to 25%.
 This gel is used in electrophoresis for the separation of proteins ranging from molecular weight
<5000 to >200,000, and polynucleotides ranges from <5 to ~ 3000 base pairs in size.

11. Polyacrylamide gels
Polyacrylamide gels are prepared by free
radical polymerization of acrylamide and a
comonomer crosslinker such as bis-
acrylamide

12. Average Pore Size of Gel Matrix

13. DNA Visualization After Gel Electrophoresis
 Ethidium Bromide
 Ethidium bromide is mixed to prepare gel before electrophoresis for uniformly dispersed
throughout the matrix.
 In the presence of ultraviolet light, ethidium bromide exhibits fluorescence. Technicians shine a
specially-calibrated UV light across the gel while a machine captures the picture of the glowing
fragments.
 Methylene Blue
 If a UV transilluminator is not available or practical, one can render DNA visible under normal
condition by soaking the finished agarose gel, with electrophoresized DNA inside, in a solution
of methylene blue overnight.
 The increased DNA stain density yields a deeper shade of blue, visible to the naked eye.
 Tracking Dyes
 Tracking dyes such as bromophenol blue and xylene cyanol can be used which move across
the aragose gel matrices during electrophoresis at the same speed as DNA.

14. Agarose Gel Electrophoresis
 A method of gel electrophoresis used in biochemistry, molecular biology, genetics,
and clinical chemistry to separate a mixed population of macromolecules such as DNA , RNA
or proteins in a matrix of agarose.
 Agarose is a natural linear polymer extracted from seaweed that forms a gel matrix by
hydrogen-bonding when heated in a buffer and allowed to cool.
 They are the most popular medium for the separation of moderate and large-sized nucleic
acids and have a wide range of separation.
 Potential difference is applied across the electrodes in a horizontal electrophoretic tank
containing agarose gel and biomolecules (such as nucleic acid or proteins) is loaded, then
molecules migrated to their respective electrodes.

15. Agarose Gel Electrophoresis
 The rate of migration of charged particles depends on the size, shape, molecular mass etc.
 In this process, 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 lights.

16. Requirement
 Instrumentation:
 An electrophoretic unit,
 A power supply,
 Gel casting trays
 Combs
 Agarose gel or media
 Electrophoresis buffer
 Composition and ionic strength of electrophoresis buffer is most important factor for the
separation of nucleic acids (DNA or RNA).
 Most routinely used buffers are:
 TAE- (Tris-acetate-EDTA), it has lower buffering capacity and generally used to separate larger
nucleic acid fragments (>12kb).

17. Requirement
 Electrophoresis buffer
 Most routinely used buffers are:
 TBE- (Tris-borate-EDTA), it has high buffering capacity and higher ionic strength and generally used for the
separation of low molecular weight compound (<1kb).
 Loading buffer: Nucleic acid is before loading on to a gel is first mixed with the gel loading buffer, which
usually consists of:-
 Salts: It creates environment with favorable ionic strength and pH of the sample, e.g., Tris-HCl.
 Metal chelator: It prevents nucleases to degrade the nucleic acid such as EDTA.
 Loading dyes: It provides color for tracking and easy monitoring of sample. Such as,
bromophenol blue, xylene cyanol.
 Transilluminator: (An ultraviolet light box), which is used to visualize bands in gels.

18. Steps Involved in AGE
 Prepare sample
 Prepare an agarose gel solution
 Gel casting
 Setting up the electrophoresis chamber
 Gel loading and sample loading
 Process of electrophoresis
 Observe the DNA
 Exposed under UV light

19. Applications of Agarose Gel Electrophoresis
 A routinely used method for separating proteins, DNA or RNA.
 Estimation of the size of DNA molecules
 Analysis of PCR products.
 Used in molecular genetic diagnosis or genetic fingerprinting.
 Separation of restricted genomic DNA prior to Southern analysis, or of RNA prior to Northern
analysis.
 Widely employed to estimate the size of DNA fragments after digesting with restriction
enzymes, e.g. in restriction mapping of cloned DNA.

20. Advantages and Disadvantages of Agarose Gel Electrophoresis
Advantages
 For most applications, only a single-component
agarose is needed and no polymerization
catalysts are required.
 Therefore, 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.

21. Polyacrylamide Gel Electrophoresis (PAGE)
 A technique widely used in biochemistry, forensic chemistry, genetics, molecular biology
and biotechnology to separate biological macromolecules, usually proteins or nucleic acids,
according to their electrophoretic mobility.
 The most commonly used form of polyacrylamide gel electrophoresis is the Sodium dodecyl
suplhate Polyacrylamide gel electrophoresis (SDS- PAGE) used mostly for the separation of
proteins.
 Polyacrylamide gels are chemically cross-linked gels formed by the polymerization of
acrylamide with a cross-linking agent, usually N,N’-methylenebisacrylamide.
 The goal of this technique is to separate a mixed sample of proteins to identify and quantify
single proteins from the mixture.
 It is also possible to use PAGE to separate DNA and RNA, but proteins are the most common
sample type.

22. Requirements for Polyacrylamide Gel Electrophoresis
 Acrylamide solutions (for resolving &
stacking gels).
 Isopropanol / distilled water.
 Gel loading buffer.
 Running buffer.
 Staining, destaining solutions.
 Protein samples
 Molecular weight markers.

23. Sample
preparation
Preparation of
polyacrylamide
gel
Electrophoresis Detection
Steps Involved in Polyacrylamide Gel Electrophoresis
 Steps Involved in Polyacrylamide Gel Electrophoresis process can be described by
dividing 4 steps:

24. Steps Involved in Polyacrylamide Gel Electrophoresis
 Sample preparation
 Samples may be any material containing proteins or nucleic acids.
 To analyze, the sample is optionally mixed with a chemical denaturant if so desired,
usually SDS for proteins or urea for nucleic acids.
 Heating the samples to at least 60 °C further promotes denaturation.
 A tracking dye may be added to the solution.
 This typically has a higher electrophoretic mobility than the analytes to allow the
experimenter to track the progress of the solution through the gel during the
electrophoretic run.

25. Sample preparation
Steps Involved in Polyacrylamide Gel Electrophoresis

26.  Preparation of polyacrylamide gel
 +
 The gels typically consist of acrylamide, bisacrylamide, produces cross-linked polymerized
structure.
 The ratio of bisacrylamide to acrylamide can be varied for special purposes, but is generally
about 1 part in 35 (1:30). 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 molecules, while
much higher percentages of acrylamide are needed to resolve smaller proteins,
 Gels are usually polymerized between two glass plates in a gel caster, with a comb inserted at
the top to create the sample wells.
 After the gel is polymerized the comb can be removed and the gel is ready for electrophoresis.
Steps Involved in Polyacrylamide Gel Electrophoresis

27. Preparation of polyacrylamide gel
Steps Involved in Polyacrylamide Gel Electrophoresis

28.  Electrophoresis
 Various buffer systems are used in PAGE depending on the nature of the sample and the
experimental objective.
 The buffers used at the anode and cathode may be the same or different.
 An electric field is applied across the gel, causing the negatively charged proteins or nucleic acids to
migrate across the gel away from the negative and towards the positive electrode (the anode).
 Depending on their size, each biomolecule moves differently through the gel matrix: small molecules
more easily fit through the pores in the gel, while larger ones have more difficulty.
 The gel is run usually for a few hours, though this depends on the voltage applied across the gel.
 After the set amount of time, the biomolecules will have migrated different distances based on their
size.
 Smaller biomolecules travel farther down the gel, while larger ones remain closer to the point of
origin.
Steps Involved in Polyacrylamide Gel Electrophoresis

29. Electrophoresis
Steps Involved in Polyacrylamide Gel Electrophoresis

30.  Detection
 Following electrophoresis, the gel may be stained (for proteins, most commonly with Coomassie
Brilliant Blue or autoradiography; for nucleic acids, ethidium bromide; or for either, silver stain),
allowing visualization of the separated proteins, or processed further (e.g. Western blot).
 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.
Steps Involved in Polyacrylamide Gel Electrophoresis

31. Applications of Polyacrylamide Gel Electrophoresis
 Measuring molecular weight.
 Peptide mapping.
 Estimation of protein size.
 Determination of protein subunits or aggregation
structures.
 Estimation of protein purity.
 Protein quantitation.
 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.

32. Advantages and Disadvantages of Polyacrylamide Gel
Electrophoresis (PAGE)
Advantages
 Stable chemically cross-linked gel
 Greater resolving power (Sharp bands)
 Can accommodate larger quantities of DNA
without significant loss in resolution
 The DNA recovered from polyacrylamide gels is
extremely pure
 The pore size of the polyacrylamide gels can be
altered in an easy and controllable fashion by
changing the concentrations of the two
monomers.
 Good for separation of low molecular weight
fragments
Disadvantages
 Generally more difficult to prepare and handle,
involving a longer time for preparation than
agarose gels.
 Toxic monomers
 Gels are tedious to prepare and often leak

33. Pulsed-field Gel Electrophoresis (PFGE)
 Pulsed-field gel electrophoresis (PFGE) is a laboratory technique used by scientists to produce a
DNA fingerprint for a bacterial isolate.
 Pulsed Field Gel Electrophoresis (PFGE) is a technique used for the separation of
large deoxyribonucleic acid (DNA) molecules by applying to a gel matrix an electric field that
periodically changes direction.
 As DNA larger than 15-20kb migrating through a gel essentially moves together in a size-
independent manner, the standard gel electrophoresis technique was unable to separate very large
molecules of DNA effectively which led to the practice of pulsed field gel electrophoresis.
 In 1982, Schwartz introduced the concept that DNA molecules larger than 50 kb can be separated
by using two alternating electric fields.

34. Principle of Pulsed Field Gel Electrophoresis (PFGE)
 While in general small fragments can find their way through the gel matrix more easily than large
DNA fragments, a threshold length exists above 30–50 kb where all large fragments will run at the
same rate, and appear in a gel as a single large diffuse band.
 However, with periodic changing of field direction, the various lengths of DNA react to the change
at differing rates.
 That is, larger pieces of DNA will be slower to realign their charge when field direction is changed,
while smaller pieces will be quicker.
 Over the course of time with the consistent changing of directions, each band will begin to
separate more and more even at very large lengths.

35. Principle of Pulsed Field Gel Electrophoresis (PFGE)
 Thus separation of very large DNA pieces using PFGE is made possible.
 The procedure for this technique is relatively similar to performing a standard gel
electrophoresis except that 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.

36. Pulsed Field Gel Electrophoresis (PFGE)

37. Steps Involved in Pulsed-field gel electrophoresis
 Lysis:
 First, the bacterial suspension is loaded into an agarose suspension.
 This is done to protect the chromosomal DNA from mechanical damage by immobilizing it into agarose blocks.
 Then the bacterial cells are lysed to release the DNA
 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.
 Analysis:
 The fragments of different organisms generated by PFGE are compared to standards manually or by computer
software like BioNumerics.

38. Steps Involved in Pulsed-field gel electrophoresis

39. Applications of Pulsed Field Gel Electrophoresis (PFGE)
 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.
 PFGE may be used for genotyping or genetic fingerprinting.
 It is commonly considered a gold standard in epidemiological studies of pathogenic
organisms.
 Subtyping has made it easier to discriminate among strains and thus to link environmental or
food isolates with clinical infections.

40. Advantages of Pulsed Field Gel Electrophoresis (PFGE)
 PFGE separates DNAs from a few kb to over 10 Mb pairs.
 Successfully applied to the subtyping of many pathogenic bacteria and has high concordance
with epidemiological relatedness.
 PFGE in the same basic format can be applied as a universal generic method for subtyping of
bacteria.
 DNA restriction patterns generated by PFGE are stable and reproducible.

41. Limitations of Pulsed Field Gel Electrophoresis (PFGE)
 Time-consuming.
 Requires trained and skilled technicians.
 Does not discriminate between all unrelated isolates.
 Don’t really know if bands of the same size are the same pieces of DNA.
 Change in one restriction site can mean more than one band change.
 Some strains cannot be typed by PFGE.