Isoelectric focusing

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ELECTROPHORESIS
Prof/ Azza abd al baky
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Electrophoresis

• Electrophoresis is the migration of charged
  molecules,particles or ion in a liquid medium
  under the influence of an electric field
• Various types defined by support used
• Paper amino acids, small peptides
• Polyacrylamide Proteins, small DNA/RNA (lt500bp)
• Agarose DNA/RNA
• Good preparative and analytical method

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From large to small and simple
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Principle

• Proteins move in the electric field. Their
  relative speed depends on the charge, size, and
  shape of the protein

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Technique of electrophoresis

• instrumentation and reagents
• (1) Two buffer boxes contain the buffer used in
  the process.
• (2) Each buffer box contains an electrode made of
  either platinum or carbon, the polarity of which
  is determined by the mode of connection to the
  power supply.
• (3)The electrophoresis support on which
  separation takes place may contact the buffer
  directly, or by means of wicks
• (4)The entire apparatus is covered to minimize
  evaporation and protect the system
• (5) The power supply to provide electrical power.

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General operations performed in conventional
electrophoresis include(1) separation (2)
staining (3) detection (4) Quantification
General Procedure
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SAMPLE APPLICATION The sample may be applied
as a spot (about 0.5 cm in diameter) or as a
uniform streak.ELECTROPHORETIC RUNThe
current is switched on after the sample has been
applied to the paper and the paper has been
equilibrated with the buffer. .The types of
buffer used depends upon the type of separation.
Once removed, the paper is dried in vacuum
oven.DETECTION AND QUANTITATIVE ASSAYTo
identify unknown components in the resolved
mixture the electrophoretogram may be compared
with another electrophoretogram on which standard
components have been electrophoresced under
identical conditions. Physical properties like
fluorescence, ultraviolet absorption or
radioactivity are exploited for detection.
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Electro-osmosis
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A simplified schematic drawing of a protein
pattern separated by cellulose acetate paper
electrophoresis
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GEL ELECTROPHORESIS

• What is a gel?
• Gel is a cross linked polymer whose composition
  and porosity is chosen based on the specific
  weight and porosity of the target molecules.
• Types of Gel
• Agarose gel.
• Polyacrylamide gel.

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Gel Electrophoresis

• Gel electrophoresis uses a cross-linked polymers
  (agarose) that contain various pores.
• Pores allow molecular sieving, where molecules
  e.g. DNA, can be separated based upon there
  mobility through the gel.

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AGAROSE GEL

• A highly purified uncharged polysaccharide
  derived from agar.
• Used to separate macromolecules such as nucleic
  acids, large proteins and protein complexes.
• It is prepared by dissolving 0.5 agarose in
  boiling water and allowing it to cool to 40C.
• It is fragile because of the formation of weak
  hydrogen bonds and hydrophobic bonds.

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POLYACRYLAMIDE GEL

• Used to separate most proteins and small
  oligonucleotides because of the presence of small
  pores.

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DNA Gel Electrophoresis

• Detection
• Dye e.g. ethidium bromide
• Audioradiography 32P,
• Blotting (see later)
• Uses
• Analytical- Can determine size of DNA fragment,
• Preparative Can identify a specific fragment
  based on size

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2D-gel (coomassie stained)

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Example of silver stained gel

• Silver staining is usually 10-100 times more
  sensitive than Coomassie Blue staining, but it is
  more complicated.
• Faint but still visible bands on this gel contain
  less than 0.5 ng of protein!

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ISOELECTRIC FOCUSING

• Electrophoretic method that separates proteins
  according to the iso-electric points
• Is ideal for seperation of amphoteric substances
• Seperation is achieved by applying a potential
  difference across a gel that contain a pH
  gradient
• Isoelectric focusing requires solid support such
  as agarose gel and polyacrylamide gel

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IEF

• Separates proteins by their isoelectric points
  (pI)
• Each protein has own pI pH at which the protein
  has equal amount of positive and negative charges
  (the net charge is zero)

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IEF example

• IEF 4-6.5 pH gradient

• Zavialov A.

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IEF

• Mixtures of ampholytes, small amphoteric
  molecules with high buffering capacity near their
  pI, are used to generate the pH gradient.
• Positively and negatively charged proteins move
  to and , respectively, until they reach pI.
• PI of proteins can be theoretically predicted.
  Therefore, IEF can also be used for protein
  identification.

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A TYPICAL ISOELECTRIC FOCUSING GEL
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Blotting Techniques

• Using specific probes that are labelled specific
  sequences of DNA can be identified.
• There are three main hybridization techniques
  which vary in the sample blotted and the probes
  used
• Northern Blot-Transfer of an RNA sample separated
  and identified using DNA or RNA probes.
• Southern Blot-Transfer of an DNA sample separated
  and identified using DNA or RNA probes.
• Western Blot- Transfer of an Protein sample
  separated and identified typically using an
  antibody.

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Blotting Techniques

• Blotting Transfer of DNA, RNA or Proteins,
  typically from a electrophoresis gel to a
  membrane e.g. nitrocellulose. This membrane can
  then be subject to further techniques such as
  hybridization.
• Hybridization Process where two complementary
  single strands of nucleic acid (DNA or RNA) form
  a double helix.

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Western Blotting (WB)

• WB is a protein detection technique that combines
  the separation power of SDS PAGE together with
  high recognition specificity of antibodies
• An antibody against the target protein could be
  purified from serum of animals (mice, rabbits,
  goats) immunized with this protein
• Alternatively, if protein contains a commonly
  used tag or epitope, an antibody against the
  tag/epitope could be purchase from a commercial
  source (e.g. anti-6 His antibody)

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WB 4 steps

• Separation of proteins using SDS PAGE
• 2. Transfer of the proteins onto e.g. a
  nitrocellulose membrane (blotting)
• 3. Immune reactions
• 4. Visualization

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WB, Step 2 Blotting
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WB, Steps 3-4 Detection
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TWO-DIMENSIONAL ELECTROPHORESIS

• This technique combines the technique IEF (first
  dimension), which separates proteins in a mixture
  according to charge (PI), with the size
  separation technique of SDS-PAGE second
  dimension).
• The combination of these two technique to give
  two-dimension(2-D)PAGE provides a highly
  sophisticated analytical method for analysing
  protein mixtures.

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• Using this method one can routinely resolve
  between 1000 and 3000 proteins from a cell or
  tissue extract and in some cases workers have
  reported the separation of between 5000 and 10000
  proteins.
• The result of this is a gel with proteins spread
  out on its surface. These proteins can then be
  detected by a variety of means, but the most
  commonly used stains are silver and coomasie
  staining.

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Capillary electrophoresis
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Capillary electrophoresis

• In CE, the classic techniques of electrophoresis
  are carried out in a small-bore, fused silica
  capillary tube, the outer diameter of such tubes
  typically varies from 180 to 375 micrometer, the
  inner diameter from 20 to 180 micrometer, and the
  total length from 20 cm up to several meters.
  This capillary tube serves as a capillary
  electrophoretic chamber that is connected to a
  detector at its terminal end and, via buffer
  reservoirs, to a high-voltage power supply
• The main advantage of CE comes from efficient
  heat dissipation compared with traditional
  electrophoresis. Improved heat dissipation
  permits the application of voltages in the range
  of 20 to 30 kV, which enhances separation
  efficiency and reduces separation time in some
  cases to less than 1 minute

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Microchip electrophoresis
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The following problems may be encountered when
peforming gel electrophoresis.

• 1. Discontinuities in sample application may be
  due to dirty applicators, which are best cleaned
  by agitating in water followed by gently pressing
  the applicators against absorbent paper. Caution
  must be used, and it is inadvisable to clean
  wires or combs by manual wiping.
• 2. Unequal migration of samples across the width
  of the gel may be due to dirty electrodes causing
  uneven application of the electrical field or to
  uneven wetting of the gel.
• 3. Distorted protein zones may be due to
• bent applicators.
• incorporation of an air bubble during sample
  application.
• over application of sample.
• excessive drying of the electrophoretic support
  before or during electrophoresis.

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• 4. Irregularities (other than broken zones)
  in sample application probably are due to
  excessively wet agarose gels. Parts of the
  applied samples may look washed out.
• Unusual bands are usually artifacts that may be
  easily recognized.
• Atypical bands in an isoenzyme pattern may be the
  result of binding by an immunoglobulin. An
  irregular, but sharp protein zone at the starting
  point that lacks the regular, somewhat diffuse
  appearance of proteins may actually be denatured
  protein resulting from a deteriorated serum.

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