electrophoresis

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electrophoresis
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BY VENKATA NAVEEN KASAGANA SWATHI SREE
KARUMURI M-PHARM -PHARMACEUTICS S.B. COLLEGE OF
PHARMACY SIVAKASI TAMIL NADU INDIA E-MAILnaveen.k
asagana_at_gmail.com
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Electrophoresis
Electro flow of electricity, phoresis, from the
Greek to carry across

• Electrophoresis is the motion of dispersed
  particles relative to a fluid under the influence
  of a spatially uniform electric field
• This electro-kinetic phenomenon was observed for
  the first time in 1807 by Reuss

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PRINCIPLE

• There are several types of electrophoresis, but
  the concepts are similar.
• The machine has an anode (positive charge) and a
  cathode (negative charge).
• Negative ions move toward the anode, and
  positive-charged ions move towards the cathode.
• The rate and distance traveled by
• these molecules help scientists
• classify and study different
• biomolecules.

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VARIOUS ELECTOPHORESIS METHODS

• Routine Electrophoresis
• High Resolution Electrophoresis (HRE)
• Gel Electrophoresis
• Agarose gel electrophoresis (AGE)
• Polyacrylamide gel Electrophoresis (PAGE)
• Isoelectric Focusing (IEF)
• Capillary Electrophoresis (CE)
• Two-Dimensional Electrophoresis
• Immunochemical Electrophoresis
• Immunofixation Electrophoresis
• Electroimmunoassay Electrophoresis
• Pulsed Field Electrophoresis

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ROUTINE ELECTROPHORESIS

• Routine electrophoresis is a generic term for the
  traditional clinical laboratory electrophoresis
  performed on a rectangle-shaped slab gel.
• Routine electrophoresis is mostly used for
  separation of proteins and has some use in
  separating nucleic acids.
• This type of electrophoresis is sometimes called
  zone electrophoresis.

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HIGH-RESOLUTION ELECTROPHORESIS (HRE)

• The technique of combining the use of agarose
  medium, a cooling system, and a modified buffer
  containing calcium ions, has become known as
  high-resolution electrophoresis (HRE).
• HRE has been applied to normal and pathological
  plasma, with the identification of as many as 14
  components that comprise more than 95 of the
  serum protein mass and have a decisive influence
  on the appearance of the electrophoretic pattern.

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• HRE (high resolution electrophoresis) is only
  possible on Cellogel and not on dry acetates. HRE
  on Cellogel is much simpler and easier than on
  agarose
• Cellogel is a film of cellulose acetate in gel
  form, produced in wet state to maintain its gel
  properties and facilitate the impregnation into
  the buffer solutions without the problem of air
  being trapped in its pores as can occur when
  using dry microporous acetate membranes

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Gel electrophoresis

• A gel is a colloid, a suspension of tiny
  particles in a medium, occurring in a solid form,
  like gelatin
• Gel electrophoresis refers to the separation of
  charged particles located in a gel when an
  electric current is applied
• Charged particles can include DNA, amino acids,
  peptides, etc

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• The gel itself is composed of either agarose or
  polyacrylamide
• Most commonly, the gel is cast in the shape of a
  thin slab, with wells for loading the sample.
• The gel is immersed within an electrophoresis
  buffer that provides ions to carry a current and
  some type of buffer to maintain the pH at a
  relatively constant value.

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Agarose gel electrophoesis(AGE)

• Agarose is a polysaccharide extracted from
  seaweed.
• It is typically used at concentrations of 0.5 to
  2.
• The higher the agarose concentration the
  "stiffer" the gel. Agarose gels are extremely
  easy to prepare. It is also non-toxic.
• Agarose gels have a large range of separation,
  but relatively low resolving power.
• By varying the concentration of agarose,
  fragments of DNA from about 200 to 50,000 bp can
  be separated using standard electrophoretic
  techniques.

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• In the case of agarose gels, pore formation is a
  physical process, resulting from a shift in
  conformation of the molecules composing it.
• In its powdered form, agarose is composed of
  loose, random coils of polysaccharide. When mixed
  with water, melted and cooled, however, these
  random coils adopt a more orderly helical
  conformation.
• However, average pore size can be controlled the
  greater the concentration of agarose in solution
  to start with, the greater the number of helices
  formed per unit of space and therefore on
  average, the smaller the pore size will be.
• Shorter molecules move faster and migrate farther
  than longer ones because shorter molecules
  migrate more easily through the pores of the gel.
  This phenomenon is called sieving .

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THE FOLLOWING ARE THE STEPS INVOLVED IN THE AGE
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Weigh out a gram of Agarose.
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• Mix the agarose with 50- 100 ml of buffer.

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• Heat to dissolve the agarose.

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• Assemble the gel tray and comb.

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Pour the gel.
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• Load one DNA sample into each well on the gel.

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Connect the gel to a low voltage power supply.
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Factors influencing

• Agarose Concentration By using gels with
  different concentrations of agarose, one can
  resolve different sizes of DNA fragments. Higher
  concentrations of agarose facilite separation of
  small DNAs, while low agarose concentrations
  allow resolution of larger DNAs.

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• Voltage
• As the voltage applied to a gel is increased,
  larger fragments migrate proportionally faster
  that small fragments. For that reason, the best
  resolution of fragments larger than about 2 kb is
  attained by applying not more than 5 volts per cm
  to the gel

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• Electrophoresis Buffer Several different buffers
  have been recommended for electrophoresis of DNA.
  
• The most commonly used for duplex DNA are TAE
  (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA).
  DNA fragments will migrate at somewhat different
  rates in these two buffers due to differences in
  ionic strength.
• Buffers not only establish a pH, but provide ions
  to support conductivity.

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PAGE polyacrylamide gel electrophoresis

• SDS-PAGE, sodium dodecyl sulfate polyacrylamide
  gel electrophoresis, is a technique widely used
  in biochemistry, forensics, genetics and
  molecular biology to separate proteins according
  to their electrophoretic mobility (a function of
  length of polypeptide chain or molecular weight).
• The purpose of this method is to separate
  proteins according to their size, and no other
  physical feature

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• Polyacrylamide is a cross-linked polymer of
  acrylamide.
• Acrylamide is a potent neurotoxin and should be
  handled with care! Wear disposable gloves when
  handling solutions of acrylamide, and a mask when
  weighing out powder.
• Polyacrylamide is considered to be non-toxic, but
  polyacrylamide gels should also be handled with
  gloves due to the possible presence of free
  acrylamide.

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Types of Polyacrylamide Gels

• There are two basic types of polyacrylamide gels
  that you will encounter in the laboratory
• Native Gels---Native Polyacrylamide gels contain
  bis and polyacrylamide (and ammonium persulfate
  and TEMED) only
• Denaturing Gels---Denaturing gels contain
  additives that maintain the molecules to be
  separated in a denatured state

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Iso - Electrofocusing

• Electrofocusing takes advantage of charge and pH
  values of proteins.
• A container is filled with a gel solution that
  has an increasing pH gradient.
• The amino acids that form polypeptides have
  different acidic or basic charges.
• The protein travels through the gel, obtaining or
  losing protons depending on its charge. As the
  protein particle moves through the gel, it
  eventually becomes neutral and gets stuck in an
  isoelectric position.

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Iso - Electrofocusing
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CAPILLARY ELECTROPHORESIS
Capillary electrophoresis is a method
similar to SDS-PAGE. It separates molecules based
on their charge and mass. Molecules are placed in
rows called capillaries filled with conductive,
electrolyte fluid. The analytes move in a speed
relative to their charge and mass. This method is
an older technique introduced in the 1960s.
SDS-PAGE is usually preferred in labs
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TWO-DIMENSIONAL GEL ELECTROPHORESIS

• Two-dimensional gel electrophoresis, abbreviated
  as 2-DE or 2-D electrophoresis, is a form of gel
  electrophoresis commonly used to analyze
  proteins. Mixtures of proteins are separated by
  two properties in two dimensions on 2D gels.
• 2-D electrophoresis begins with 1-D
  electrophoresis but then separates the molecules
  by a second property in a direction 90 degrees
  from the first. In 1-D electrophoresis, proteins
  (or other molecules) are separated in one
  dimension, so that all the proteins/molecules
  will lie along a lane but that the molecules are
  spread out across a 2-D gel. Because it is
  unlikely that two molecules will be similar in
  two distinct properties, so molecules are more
  effectively separated in 2-D electrophoresis than
  in 1-D electrophoresis.

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Affinity electrophoresis

• Affinity electrophoresis is a general name for
  many analytical methods used in biochemistry and
  biotechnology. Both qualitative and quantitative
  information may be obtained through affinity
  electrophoresis. The methods include the
  so-called mobility shift electrophoresis, charge
  shift electrophoresis and affinity capillary
  electrophoresis.

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• The methods are based on changes in the
  electrophoretic pattern of molecules (mainly
  macromolecules) through biospecific interaction
  or complex formation. The interaction or binding
  of a molecule, charged or uncharged, will
  normally change the electrophoretic properties of
  a molecule. Membrane proteins may be identified
  by a shift in mobility induced by a charged
  detergent. Nucleic acids or nucleic acid
  fragments may be characterized by their affinity
  to other molecules.

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Pulsed Field - Electrophoresis

• Pulsed Field - any electrophoresis process that
  uses more than one electric field alternatingly.
• PFGE allows investigators to separate much larger
  pieces of DNA than conventional agarose gel
  electrophoresis. In conventional gels, the
  current is applied in a single direction.

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The red-striped arrows represent the direction
of the current.  
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Example of a real PFGE drug resistant
Staphylococcus aureus. The molecular weight
markers are digested lambda phage (?) and are
given in kb. a
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Applications

• Electrophoresis has been used as a means of
  identifying the genetic defects that cause many
  hereditary diseases .PFGE
• It may be used for genotyping or genetic
  fingerprinting.
• It is commonly considered a gold standard in
  epidemiological studies of pathogenic organisms.

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• Pharmaceuticals
• Inorganic ions
• Industrial chemicals
• Chiral drugs
• Carbohydrates
• Foodstuffs
• Plants extracts
• Oligonucleotides

• Amino acids
• Peptides
• Proteins
• PCR products
• DNA fragments
• RNA
• Bacteria
• Viruses

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• REFERENCES
• ELECTROPHORESIS BY MAUREEN MELVIN
• AUTHOR STREAM .COM
• SLIDEWORLD.COM
• WIKIPEDIA.ORG

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THANK YOU