SlideShare a Scribd company logo

Gel Electrophoresis

contains introduction on Gel eletrophoresis,principle, its types and applications of Gel electrophoresis in compact manner.

1 of 27
1
Introduction
• Positive or negative electrical charges are frequently
associated with biomolecules.
• When placed in an electric field, charged biomolecules
move towards the electrode of opposite charge due to
the phenomenon of electrostatic attraction.
• Electrophoresis is the separation of charged molecules
in an applied electric field.
• The relative mobility of individual molecules depends
on several factors such as net charge, charge/mass
ratio,molecular shape and the temperature, porosity
and viscosity of the matrix.
• Complex mixtures can be separated to very high
resolution by this process.
2
Principle of Electrophoresis
If a mixture of electrically charged biomolecules is
placed in an electric field of field strength
E, they will freely move towards the electrode of
opposite charge. However, different
molecules will move at quite different and
individual rates depending on the physical
characteristics of the molecule and on
experimental system used. The velocity or
movement, ν, of a charged molecule in an electric
field depends on variables described by
ν= E.q / f (1)
where f is the frictional coefficient and q is the
net charge on the molecule
3
The frictional coefficient describes frictional
resistance to mobility and depends on a
number of factors such as mass of the
molecule, its degree of compactness, buffer
viscosity and the porosity of the matrix in
which the experiment is performed. The net
charge is determined by the number of
positive and negative charges in the molecule.
Charges are conferred on proteins by amino
acid side chains as well as by groups arising
from post translational modifications such as
deamidation, acylation or phosphorylation.
4
Gel electrophoresis
Hydrated gel networks have many desirable properties for
electrophoresis. They allow a wide variety of mechanically
stable experimental formats such as horizontal/vertical
electrophoresis in slab gels or electrophoresis in tubes or
capillaries. The mechanical stability also facilitates post
electrophoretic manipulation making further experimentation
possible such as blotting, electro-elution or MS identification
/finger printing of intact proteins or of proteins digested in gel
slices. Since gels used in biochemistry are chemically rather
unreactive, they interact minimally with biomolecules during
electrophoresis allowing separation based on physical rather
than chemical differences between sample components
5
6

More Related Content

What's hot

Zone electrophoresis and its types
Zone electrophoresis and its typesZone electrophoresis and its types
Zone electrophoresis and its typesniharika Sola
 
ISOELECTRIC FOCUSING PPT - SLIDE SHARE
ISOELECTRIC FOCUSING PPT - SLIDE SHAREISOELECTRIC FOCUSING PPT - SLIDE SHARE
ISOELECTRIC FOCUSING PPT - SLIDE SHAREAditi Chaturvedi
 
Electrophoresis ppt.
Electrophoresis ppt.Electrophoresis ppt.
Electrophoresis ppt.gulamrafey
 
Capillary Electrophoresis
Capillary ElectrophoresisCapillary Electrophoresis
Capillary ElectrophoresisSantoshi10
 
Techniques of electrophoresis
Techniques of electrophoresisTechniques of electrophoresis
Techniques of electrophoresisSayanti Sau
 
Moving boundary electrophoresis mpat
Moving boundary electrophoresis mpatMoving boundary electrophoresis mpat
Moving boundary electrophoresis mpatHarish Rahar
 
Introduction, Principle, Instrumentation and Applications of SDS-PAGE
Introduction, Principle, Instrumentation and Applications of SDS-PAGEIntroduction, Principle, Instrumentation and Applications of SDS-PAGE
Introduction, Principle, Instrumentation and Applications of SDS-PAGEMohammed Mubeen
 
Isoelectric focussing
Isoelectric focussingIsoelectric focussing
Isoelectric focussingarushe143
 
Gel electrophoresis himanshu
Gel electrophoresis himanshuGel electrophoresis himanshu
Gel electrophoresis himanshuhimanshu kamboj
 
Paper electrophoresis
Paper electrophoresis Paper electrophoresis
Paper electrophoresis PUNEET NIRMAL
 
GEL ELECTROPHORESIS
GEL ELECTROPHORESISGEL ELECTROPHORESIS
GEL ELECTROPHORESISmoazzam99
 
Capillary electrophoresis principles and applications
Capillary electrophoresis principles and applications   Capillary electrophoresis principles and applications
Capillary electrophoresis principles and applications Indira Shastry
 
Isoelectric focusing
Isoelectric focusingIsoelectric focusing
Isoelectric focusingPalviSingla2
 

What's hot (20)

Gel Filtration
Gel FiltrationGel Filtration
Gel Filtration
 
Zone electrophoresis and its types
Zone electrophoresis and its typesZone electrophoresis and its types
Zone electrophoresis and its types
 
Electrophoresis
ElectrophoresisElectrophoresis
Electrophoresis
 
ISOELECTRIC FOCUSING PPT - SLIDE SHARE
ISOELECTRIC FOCUSING PPT - SLIDE SHAREISOELECTRIC FOCUSING PPT - SLIDE SHARE
ISOELECTRIC FOCUSING PPT - SLIDE SHARE
 
Electrophoresis ppt.
Electrophoresis ppt.Electrophoresis ppt.
Electrophoresis ppt.
 
Paper electrophoresis
Paper electrophoresisPaper electrophoresis
Paper electrophoresis
 
Capillary Electrophoresis
Capillary ElectrophoresisCapillary Electrophoresis
Capillary Electrophoresis
 
Slab gel electrophoresis
Slab gel electrophoresisSlab gel electrophoresis
Slab gel electrophoresis
 
Techniques of electrophoresis
Techniques of electrophoresisTechniques of electrophoresis
Techniques of electrophoresis
 
Moving boundary electrophoresis mpat
Moving boundary electrophoresis mpatMoving boundary electrophoresis mpat
Moving boundary electrophoresis mpat
 
Gel electrophoresis
Gel electrophoresisGel electrophoresis
Gel electrophoresis
 
Introduction, Principle, Instrumentation and Applications of SDS-PAGE
Introduction, Principle, Instrumentation and Applications of SDS-PAGEIntroduction, Principle, Instrumentation and Applications of SDS-PAGE
Introduction, Principle, Instrumentation and Applications of SDS-PAGE
 
Isoelectric focussing
Isoelectric focussingIsoelectric focussing
Isoelectric focussing
 
Gel electrophoresis himanshu
Gel electrophoresis himanshuGel electrophoresis himanshu
Gel electrophoresis himanshu
 
Capillary electrophoresis
Capillary electrophoresisCapillary electrophoresis
Capillary electrophoresis
 
Paper electrophoresis
Paper electrophoresis Paper electrophoresis
Paper electrophoresis
 
GEL ELECTROPHORESIS
GEL ELECTROPHORESISGEL ELECTROPHORESIS
GEL ELECTROPHORESIS
 
Capillary electrophoresis principles and applications
Capillary electrophoresis principles and applications   Capillary electrophoresis principles and applications
Capillary electrophoresis principles and applications
 
Isoelectric focusing
Isoelectric focusingIsoelectric focusing
Isoelectric focusing
 
Electrophoresis
ElectrophoresisElectrophoresis
Electrophoresis
 

Similar to Gel Electrophoresis

Similar to Gel Electrophoresis (20)

Electrophoresis and various techniques
Electrophoresis and various techniquesElectrophoresis and various techniques
Electrophoresis and various techniques
 
Electrophoresis
ElectrophoresisElectrophoresis
Electrophoresis
 
ELECTRPOPHOROSIS
ELECTRPOPHOROSISELECTRPOPHOROSIS
ELECTRPOPHOROSIS
 
Electrophoresis-PAPER ELECTROPHORESIS,GEL ELCTROPHORESIS, PAGE-SDS AND NON-SDS
Electrophoresis-PAPER ELECTROPHORESIS,GEL ELCTROPHORESIS, PAGE-SDS AND NON-SDSElectrophoresis-PAPER ELECTROPHORESIS,GEL ELCTROPHORESIS, PAGE-SDS AND NON-SDS
Electrophoresis-PAPER ELECTROPHORESIS,GEL ELCTROPHORESIS, PAGE-SDS AND NON-SDS
 
Gel electrophoresis
Gel electrophoresisGel electrophoresis
Gel electrophoresis
 
Electrophoresis
ElectrophoresisElectrophoresis
Electrophoresis
 
Electrophoresis
Electrophoresis Electrophoresis
Electrophoresis
 
Electrophoresis
ElectrophoresisElectrophoresis
Electrophoresis
 
Separation of Biomolecules
Separation of BiomoleculesSeparation of Biomolecules
Separation of Biomolecules
 
Electrophoresis
ElectrophoresisElectrophoresis
Electrophoresis
 
Electrophoresis
Electrophoresis Electrophoresis
Electrophoresis
 
Global analysis of protein by kk sahu sir
Global analysis of protein by kk sahu sirGlobal analysis of protein by kk sahu sir
Global analysis of protein by kk sahu sir
 
electrophoresis
electrophoresiselectrophoresis
electrophoresis
 
electrophoresis analysis ,.pptx
electrophoresis analysis ,.pptxelectrophoresis analysis ,.pptx
electrophoresis analysis ,.pptx
 
electrophoresis
electrophoresiselectrophoresis
electrophoresis
 
Electrophoresis & its types
Electrophoresis & its typesElectrophoresis & its types
Electrophoresis & its types
 
219159 lecture 10
219159 lecture 10219159 lecture 10
219159 lecture 10
 
Electrophoresis
ElectrophoresisElectrophoresis
Electrophoresis
 
Electrophoresis
ElectrophoresisElectrophoresis
Electrophoresis
 
gel electrophoresis
gel electrophoresisgel electrophoresis
gel electrophoresis
 

Recently uploaded

Cytotoxic Activity of Linum usitatissimum L. Essential oil against Lung Adeno...
Cytotoxic Activity of Linum usitatissimum L. Essential oil against Lung Adeno...Cytotoxic Activity of Linum usitatissimum L. Essential oil against Lung Adeno...
Cytotoxic Activity of Linum usitatissimum L. Essential oil against Lung Adeno...AmalDhivaharS
 
Culture methods ^J preparation and sterilization of media.pptx
Culture methods ^J preparation and sterilization of media.pptxCulture methods ^J preparation and sterilization of media.pptx
Culture methods ^J preparation and sterilization of media.pptxjipaga3672
 
PSILOTUM : structure, morphology, anatomy, reproduction , life cycle etc.
PSILOTUM : structure, morphology, anatomy,  reproduction , life cycle etc.PSILOTUM : structure, morphology, anatomy,  reproduction , life cycle etc.
PSILOTUM : structure, morphology, anatomy, reproduction , life cycle etc.Silpa Selvaraj
 
Safety_and_Health_Programs_v-03-01-17.pptx
Safety_and_Health_Programs_v-03-01-17.pptxSafety_and_Health_Programs_v-03-01-17.pptx
Safety_and_Health_Programs_v-03-01-17.pptxmohamedsami2233
 
dkNET Webinar: The Collaborative Microbial Metabolite Center – Democratizing ...
dkNET Webinar: The Collaborative Microbial Metabolite Center – Democratizing ...dkNET Webinar: The Collaborative Microbial Metabolite Center – Democratizing ...
dkNET Webinar: The Collaborative Microbial Metabolite Center – Democratizing ...dkNET
 
CHEMICAL TESTS FOR GLYCOSIDES AND ALKALOIDS.pptx
CHEMICAL TESTS FOR GLYCOSIDES AND ALKALOIDS.pptxCHEMICAL TESTS FOR GLYCOSIDES AND ALKALOIDS.pptx
CHEMICAL TESTS FOR GLYCOSIDES AND ALKALOIDS.pptxASWIN ANANDH
 
Seminario biología molecular Lina Charris
Seminario biología molecular Lina CharrisSeminario biología molecular Lina Charris
Seminario biología molecular Lina CharrisLinaMarcelaCharrisRa
 
electrophoresis: types, advantages, disadvantages and applications.
electrophoresis: types, advantages, disadvantages and applications.electrophoresis: types, advantages, disadvantages and applications.
electrophoresis: types, advantages, disadvantages and applications.Silpa Selvaraj
 
Introduction to Chromatography (Column chromatography)
Introduction to Chromatography (Column chromatography)Introduction to Chromatography (Column chromatography)
Introduction to Chromatography (Column chromatography)Ahmed Metwaly
 
1.0 - The Light Miscroscope.ppt microscopy
1.0 - The Light Miscroscope.ppt microscopy1.0 - The Light Miscroscope.ppt microscopy
1.0 - The Light Miscroscope.ppt microscopystephenopokuasante
 
green chemistry, clean sustainable environment.ppt
green chemistry, clean sustainable environment.pptgreen chemistry, clean sustainable environment.ppt
green chemistry, clean sustainable environment.pptRashmiSanghi1
 
Presentacion Mariana Arango- biología molecular
Presentacion Mariana Arango- biología molecularPresentacion Mariana Arango- biología molecular
Presentacion Mariana Arango- biología molecularmarianaarangop
 
Open Access Publishing in Astrophysics and the Open Journal of Astrophysics
Open Access Publishing in Astrophysics and the Open Journal of AstrophysicsOpen Access Publishing in Astrophysics and the Open Journal of Astrophysics
Open Access Publishing in Astrophysics and the Open Journal of AstrophysicsPeter Coles
 
discussion on the endocrine system for science grade10.pptx
discussion on the endocrine system for science grade10.pptxdiscussion on the endocrine system for science grade10.pptx
discussion on the endocrine system for science grade10.pptxShePerezDelaCruz
 
Anti-Obesity Activity of Anthocyanins and Corresponding Introduction in Dieta...
Anti-Obesity Activity of Anthocyanins and Corresponding Introduction in Dieta...Anti-Obesity Activity of Anthocyanins and Corresponding Introduction in Dieta...
Anti-Obesity Activity of Anthocyanins and Corresponding Introduction in Dieta...AmalDhivaharS
 
Ento-322, Agrochemicals for agriculture usee
Ento-322, Agrochemicals for agriculture useeEnto-322, Agrochemicals for agriculture usee
Ento-322, Agrochemicals for agriculture useeDrAnita Sharma
 
Anatomy-Reproductive-System for grade 10
Anatomy-Reproductive-System for grade 10Anatomy-Reproductive-System for grade 10
Anatomy-Reproductive-System for grade 10rolanaribato30
 
Weak-lensing detection of intracluster filaments in the Coma cluster
Weak-lensing detection of intracluster filaments in the Coma clusterWeak-lensing detection of intracluster filaments in the Coma cluster
Weak-lensing detection of intracluster filaments in the Coma clusterSérgio Sacani
 
commercial production of cellulase enzyme and its uses
commercial production of cellulase enzyme and its usescommercial production of cellulase enzyme and its uses
commercial production of cellulase enzyme and its usesSilpa Selvaraj
 

Recently uploaded (20)

Cytotoxic Activity of Linum usitatissimum L. Essential oil against Lung Adeno...
Cytotoxic Activity of Linum usitatissimum L. Essential oil against Lung Adeno...Cytotoxic Activity of Linum usitatissimum L. Essential oil against Lung Adeno...
Cytotoxic Activity of Linum usitatissimum L. Essential oil against Lung Adeno...
 
Culture methods ^J preparation and sterilization of media.pptx
Culture methods ^J preparation and sterilization of media.pptxCulture methods ^J preparation and sterilization of media.pptx
Culture methods ^J preparation and sterilization of media.pptx
 
PSILOTUM : structure, morphology, anatomy, reproduction , life cycle etc.
PSILOTUM : structure, morphology, anatomy,  reproduction , life cycle etc.PSILOTUM : structure, morphology, anatomy,  reproduction , life cycle etc.
PSILOTUM : structure, morphology, anatomy, reproduction , life cycle etc.
 
Safety_and_Health_Programs_v-03-01-17.pptx
Safety_and_Health_Programs_v-03-01-17.pptxSafety_and_Health_Programs_v-03-01-17.pptx
Safety_and_Health_Programs_v-03-01-17.pptx
 
dkNET Webinar: The Collaborative Microbial Metabolite Center – Democratizing ...
dkNET Webinar: The Collaborative Microbial Metabolite Center – Democratizing ...dkNET Webinar: The Collaborative Microbial Metabolite Center – Democratizing ...
dkNET Webinar: The Collaborative Microbial Metabolite Center – Democratizing ...
 
REGULATION OF METABOLISM IN PLANTS AND THE DIFFERENT MECHANISMS
REGULATION OF METABOLISM IN PLANTS  AND THE DIFFERENT MECHANISMSREGULATION OF METABOLISM IN PLANTS  AND THE DIFFERENT MECHANISMS
REGULATION OF METABOLISM IN PLANTS AND THE DIFFERENT MECHANISMS
 
CHEMICAL TESTS FOR GLYCOSIDES AND ALKALOIDS.pptx
CHEMICAL TESTS FOR GLYCOSIDES AND ALKALOIDS.pptxCHEMICAL TESTS FOR GLYCOSIDES AND ALKALOIDS.pptx
CHEMICAL TESTS FOR GLYCOSIDES AND ALKALOIDS.pptx
 
Seminario biología molecular Lina Charris
Seminario biología molecular Lina CharrisSeminario biología molecular Lina Charris
Seminario biología molecular Lina Charris
 
electrophoresis: types, advantages, disadvantages and applications.
electrophoresis: types, advantages, disadvantages and applications.electrophoresis: types, advantages, disadvantages and applications.
electrophoresis: types, advantages, disadvantages and applications.
 
Introduction to Chromatography (Column chromatography)
Introduction to Chromatography (Column chromatography)Introduction to Chromatography (Column chromatography)
Introduction to Chromatography (Column chromatography)
 
1.0 - The Light Miscroscope.ppt microscopy
1.0 - The Light Miscroscope.ppt microscopy1.0 - The Light Miscroscope.ppt microscopy
1.0 - The Light Miscroscope.ppt microscopy
 
green chemistry, clean sustainable environment.ppt
green chemistry, clean sustainable environment.pptgreen chemistry, clean sustainable environment.ppt
green chemistry, clean sustainable environment.ppt
 
Presentacion Mariana Arango- biología molecular
Presentacion Mariana Arango- biología molecularPresentacion Mariana Arango- biología molecular
Presentacion Mariana Arango- biología molecular
 
Open Access Publishing in Astrophysics and the Open Journal of Astrophysics
Open Access Publishing in Astrophysics and the Open Journal of AstrophysicsOpen Access Publishing in Astrophysics and the Open Journal of Astrophysics
Open Access Publishing in Astrophysics and the Open Journal of Astrophysics
 
discussion on the endocrine system for science grade10.pptx
discussion on the endocrine system for science grade10.pptxdiscussion on the endocrine system for science grade10.pptx
discussion on the endocrine system for science grade10.pptx
 
Anti-Obesity Activity of Anthocyanins and Corresponding Introduction in Dieta...
Anti-Obesity Activity of Anthocyanins and Corresponding Introduction in Dieta...Anti-Obesity Activity of Anthocyanins and Corresponding Introduction in Dieta...
Anti-Obesity Activity of Anthocyanins and Corresponding Introduction in Dieta...
 
Ento-322, Agrochemicals for agriculture usee
Ento-322, Agrochemicals for agriculture useeEnto-322, Agrochemicals for agriculture usee
Ento-322, Agrochemicals for agriculture usee
 
Anatomy-Reproductive-System for grade 10
Anatomy-Reproductive-System for grade 10Anatomy-Reproductive-System for grade 10
Anatomy-Reproductive-System for grade 10
 
Weak-lensing detection of intracluster filaments in the Coma cluster
Weak-lensing detection of intracluster filaments in the Coma clusterWeak-lensing detection of intracluster filaments in the Coma cluster
Weak-lensing detection of intracluster filaments in the Coma cluster
 
commercial production of cellulase enzyme and its uses
commercial production of cellulase enzyme and its usescommercial production of cellulase enzyme and its uses
commercial production of cellulase enzyme and its uses
 

Gel Electrophoresis

  • 1. 1
  • 2. Introduction • Positive or negative electrical charges are frequently associated with biomolecules. • When placed in an electric field, charged biomolecules move towards the electrode of opposite charge due to the phenomenon of electrostatic attraction. • Electrophoresis is the separation of charged molecules in an applied electric field. • The relative mobility of individual molecules depends on several factors such as net charge, charge/mass ratio,molecular shape and the temperature, porosity and viscosity of the matrix. • Complex mixtures can be separated to very high resolution by this process. 2
  • 3. Principle of Electrophoresis If a mixture of electrically charged biomolecules is placed in an electric field of field strength E, they will freely move towards the electrode of opposite charge. However, different molecules will move at quite different and individual rates depending on the physical characteristics of the molecule and on experimental system used. The velocity or movement, ν, of a charged molecule in an electric field depends on variables described by ν= E.q / f (1) where f is the frictional coefficient and q is the net charge on the molecule 3
  • 4. The frictional coefficient describes frictional resistance to mobility and depends on a number of factors such as mass of the molecule, its degree of compactness, buffer viscosity and the porosity of the matrix in which the experiment is performed. The net charge is determined by the number of positive and negative charges in the molecule. Charges are conferred on proteins by amino acid side chains as well as by groups arising from post translational modifications such as deamidation, acylation or phosphorylation. 4
  • 5. Gel electrophoresis Hydrated gel networks have many desirable properties for electrophoresis. They allow a wide variety of mechanically stable experimental formats such as horizontal/vertical electrophoresis in slab gels or electrophoresis in tubes or capillaries. The mechanical stability also facilitates post electrophoretic manipulation making further experimentation possible such as blotting, electro-elution or MS identification /finger printing of intact proteins or of proteins digested in gel slices. Since gels used in biochemistry are chemically rather unreactive, they interact minimally with biomolecules during electrophoresis allowing separation based on physical rather than chemical differences between sample components 5
  • 6. 6
  • 7. Gel types In general the macromolecules solution is electrophoresed through some kind of matrix. The matrix acts as a molecular sieve to aid in the separation of molecules on the basis of size. The kind of supporting matrix used depends on the type of molecules to be separated and on the desired basis for separation: charge, molecular weight or both (Dolnik, V.; 1997). The most commonly used materials for the separation of nucleic acids and proteins are agarose and acrylamide. 7
  • 8. Table 1. above shows some media used for Gel Electrophoresis Agarose: The most widely used polysaccharide gel matrix nowadays is that formed with agarose. This is a polymer composed of a repeating disaccharide unit called agarobiose which consists of galactose and 3,6- anhydrogalactose (Fig. 1). Agarose gives a more uniform degree of porosity than starch and this may be varied by altering the starting concentration of the suspension (low concentrations give large pores while high concentrations give smaller pores). This gel has found wide spread use especially in the separation of DNA molecules LIMITATION: limited mechanical stability 8
  • 9. Acrylamide: A far stronger gel suitable for electrophoretic separation of both proteins and nucleic acids, may be formed by the polymerization of acrylamide. The inclusion of a small amount of acrylamide cross linked by a methylene bridge (N,N′ methylene bisacrylamide) allows formation of a cross linked gel with a highly-controlled porosity which is also mechanically strong and chemically inert. 9
  • 10. . The polymerization reaction is initiated by persulphate radicals and catalyzed by N,N,N’,N’-tetramethylethylendiamine(TEMED). 10
  • 11. Staining of gel One of the most important aspects of gel electrophoresis technique is staining. Once sample molecules have separated in the gel matrix it is necessary to visualize their position. This is achieved by staining with an agent appropriate for the sample. Some of the more common staining methods used in biochemistry are listed in Table below. 11
  • 12. 12
  • 13. Preparation and running of standard agarose gels The equipment and supplies necessary for conducting agarose gel electrophoresis are relatively simple and include: 1.An electrophoresis chamber and power supply 2.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. 3.Sample combs, around which molten medium is poured to form sample wells in the gel. 4.Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris- borate-EDTA (TBE). 5.Loading buffer, which 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 the gel and allow visual monitoring or how far the electrophoresis has proceeded. 13
  • 14. 14
  • 15. Staining: DNA molecules are easily visualized under an ultraviolet lamp when electrophoresed in the presence of ethidium bromide. Alternatively, nucleic acids can be stained after electrophoretic separation by soaking the gel in a solution of ethidium bromide. NOTE: Ethidium bromide is a known mutagen and should be handled as a hazardous chemical - wear gloves while handling 15
  • 16. Transilluminator (an ultraviolet light box), which is used to visualize stained DNA in gels. 16
  • 17. Types of Gel Electrophoresis 17
  • 18. SDS-PAGE Electrophoresis • SDS-PAGE is an analytical technique to separate proteins based on their molecular weight. separates proteins primarily by mass. • Non denaturing PAGE, also called native PAGE, separates proteins according to their mass:charge ratio. 18
  • 19. When proteins are separated by electrophoresis through a gel matrix, smaller proteins migrate faster due to less resistance from the gel matrix. Other influences on the rate of migration through the gel matrix include the structure and charge of the proteins. In SDS-PAGE, the use of sodium dodecyl sulfate (SDS, also known as sodium lauryl sulfate) and polyacrylamide gel largely eliminates the influence of the structure and charge, and proteins are separated solely based on polypeptide chain length. SDS is a detergent with a strong protein-denaturing effect and binds to the protein backbone at a constant molar ratio. In the presence of SDS and a reducing agent that cleaves disulfide bonds critical for proper folding, proteins unfold into linear chains with negative charge proportional to the polypeptide chain length 19
  • 20. 20
  • 21. 21
  • 22. NATIVE PAGE • Native page electrophoresis is run in absence of SDS. • Here, the mobility depends upon both the protein’s charge and its hydrodynamic size. • If native page is carried out near neutral pH to avoid acid or alkaline denaturation. • Then it can be used to study confirmation, self association or aggregation . ADVANTAGE: It is possible to recover proteins in their native state after the separation. 22
  • 23. ISOELECTRIC FOCUSING • It is a technique for separating different molecules by differences in their isoelectric point. • It is a type of zone electrophoresis which is performed on proteins in a gel. • Separation is achieved by applying a potential difference across a gel that contains a pH gradient. • It requires a solid support such as agarose gel and polyacrylamide gel • All proteins have isoelectric point pH. • Proteins migrate to the point where net charge is zero. 23
  • 24. Applications 1. Gel electrophoresis is used in forensics, molecular biology, genetics, microbiology and biogenetics. 2. Estimation of size of DNA molecules following restriction enzymes digestion e.g. In restriction mapping of cloned DNA. 3. Analysis of lipoproteins. 4. Used in DNA fingerprinting. 5. Used for DNA sequencing. 6. separation of organicacids,alkaloids,carbohydrates , amino acids and nucleic acids 24
  • 25. 7.Used in food industry. 8. Used for separating Protein mixtures. 9. Separation of serum proteins. 25
  • 26. BIBLIOGRAPHY • https://www.intechopen.com/books/gel-electrophoresis-principles- and-basics/gel-electrophoresis-and-its-applications • https://www.khanacademy.org/science/biology/biotech-dna- technology/dna-sequencing-pcr-electrophoresis/a/gel- electrophoresis • http://chemfuuast.weebly.com/uploads/1/2/8/9/12894433/presen tation_on_electrophoresis_by_dr_shah_ali-ul-qadir.pdf • https://biologydictionary.net/gel-electrophoresis/ • http://ruo.mbl.co.jp/bio/g/support/method/sds-page.html • https://www.slideshare.net/udayarajitha/gel-electrophoresis- 64123389?qid=6d6a4a18-4530-4360-8b2b- 61cbcd1dd785&v=&b=&from_search=2 • https://www.slideshare.net/mbn1994/introduction-principle- instrumentation-and-applications-of-sdspage-55728195 26
  • 27. 27