Introduction
Gel Electrophoresis
Principle of separation
Instrument and reagents
Factors affecting separation in gel electrophoresis
Applications
Electrophoresis apparatus
Buffer
Power supply
Supporting media
Detection and Quantification
Agarose
Polyacrylamide
Separation is brought about through molecular sieving technique, based on the molecular size of the substances. Gel material acts as a "molecular sieve”.
Gel is a colloid in a solid form (99% is water).
It is important that the support media is electrically neutral.
Different types of gels which can be used are; Agar and Agarose gel, Starch, Sephadex, Polyacrylamide gels.
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.
Introduction
Gel Electrophoresis
Principle of separation
Instrument and reagents
Factors affecting separation in gel electrophoresis
Applications
Electrophoresis apparatus
Buffer
Power supply
Supporting media
Detection and Quantification
Agarose
Polyacrylamide
Separation is brought about through molecular sieving technique, based on the molecular size of the substances. Gel material acts as a "molecular sieve”.
Gel is a colloid in a solid form (99% is water).
It is important that the support media is electrically neutral.
Different types of gels which can be used are; Agar and Agarose gel, Starch, Sephadex, Polyacrylamide gels.
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.
Introduction, Principle, Instrumentation and Applications of SDS-PAGEMohammed Mubeen
The following presentation contains helpful information regarding SDS-PAGE, including the history, introduction, principle, instrumentation, advantages and applications of SDS-PAGE.
This presentation contain the information about gel electrophoresis method , instruments & types.
Electrophoresis is a method through biological molecules are separated by applying an electric field.
Main purpose of this method is to determine the number , amount & mobility of biological component.
There are some internal & external factors that affects the process of electrophoresis.
The bio-molecules have charge on it & when we apply an electric field , the charge particles move to the opposite cathode. In this way, charge particles are separated
There are 3 types of gels that use in this process .
In this buffers are also used which provide ions that carry a current.
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
In this slide contains principle, types, materials used, factors affecting gel electrophoresis.
Presented by: I. Sai Reddemma (Department of pharmacology).
RIPER, anantapur.
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.
PAGE is a subtype of the gel electrophoresis whereby the normal gel is replaced with polyacrylamide gels use as the support matrix.
widely used and has very much importance.
COMPLETE PROCEDURE & USES are described in the slide.
Introduction, Principle, Instrumentation and Applications of SDS-PAGEMohammed Mubeen
The following presentation contains helpful information regarding SDS-PAGE, including the history, introduction, principle, instrumentation, advantages and applications of SDS-PAGE.
This presentation contain the information about gel electrophoresis method , instruments & types.
Electrophoresis is a method through biological molecules are separated by applying an electric field.
Main purpose of this method is to determine the number , amount & mobility of biological component.
There are some internal & external factors that affects the process of electrophoresis.
The bio-molecules have charge on it & when we apply an electric field , the charge particles move to the opposite cathode. In this way, charge particles are separated
There are 3 types of gels that use in this process .
In this buffers are also used which provide ions that carry a current.
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
In this slide contains principle, types, materials used, factors affecting gel electrophoresis.
Presented by: I. Sai Reddemma (Department of pharmacology).
RIPER, anantapur.
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.
PAGE is a subtype of the gel electrophoresis whereby the normal gel is replaced with polyacrylamide gels use as the support matrix.
widely used and has very much importance.
COMPLETE PROCEDURE & USES are described in the slide.
it is a short ppt. on Electrophoresis. Which gives it's defination, Types of electrophoresis ; Like:- Zone electrophoresis, Isoelectric focussing, Immunoelectrophoresis. and their images.
Gel Electrophoresis, ITS FACTOR AFFECTING, ITS TYPES,NORMAL METHODOLOGY, PAGERitamMukherjee11
ELECTROPHORESIS may be defined as the migration of the charged particle through a solution under the influence of an external electric field.
GEL ELECTROPHORESIS is a technique used to separate biomacromolecules (such as DNA, RNA, proteins, etc.) and their fragments based on their size and charge, by applying an electric field to a gel with small pores.
INTRODUCTION - Separation is brought about through molecular sieving techniques, based on the molecular size of the substances. Gel material acts as a “ molecular sieve”
It is important that support media is electrically neutral.
There are different types of gel that can be used, they are Agarose, Polyacrylamide, Starch, and Sephadex.
A porous gel acts as a sieve by retarding or, in some cases by completely obstructing the movement of macromolecules which allowed smaller molecules to migrate freely.
PRINCIPLE - According to charge: When charged molecules are placed in an electric field, they migrate toward either the positive (anode) or negative (cathode) pole according to their charge.
According to size: The smaller molecules move more swiftly than the larger-sized ones, as they can travel through the pores more easily than the later.
According to Molecular weight: The smaller molecular weight will move faster than the larger molecular weight compound.
Sepration of molecules on the basis of applied Electric Field
Categorized into 1) Zone Electrophoresis 2) Moving Boundary Electrophoresis
We can seprate macromolecules (DNA , RNA, PROTEINS )on the basis of their charge, size shape & molecular weight
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.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
This presentation explores a brief idea about the structural and functional attributes of nucleotides, the structure and function of genetic materials along with the impact of UV rays and pH upon them.
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
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.
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
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
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
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
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
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