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ELECTROPHORETIC METHODS
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Basic principles of electrophoresis

• It is the process of moving charged biomolecules
  in solution by applying an electrical field
  across the mixture.
• Biomolecules moved with a speed dependent on
  their charge, shape, and size and separation
  occures on the basis of molecular size.
• Electrophoresis is used for analysis and
  purification of very large molecules (proteins,
  nucleic acids)
• for analysis of simpler charged molecules
  (sugars, amino acids, peptides, nucleotides, and
  simpler ions).

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• When charged molecules are placed in an electric
  field, they migrate toward either the positive
  (anode) or negative (cathode) pole according to
  their charge.
• Factors influenced electrophoresis mobility
• net charge of the molecule
• size and shape
• concentration of the molecule in solution

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Amino acids have characteristic titration curves
Proton donor
Proton acceptor
At the midpoint pK9.60 there is equimolar
concentration of proton donor and proton acceptor.
Izoelectric point
Dipolar ion
At the midpoint pK12.34 there is equimolar
concentration of proton donor and proton acceptor.
Proton donor
Proton acceptor
Fully protonated form at wery low pH
Adopted from D.L. Nelson, M.M. Cox Lehninger
Principle of Biochemistry
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• Electrophoresis is carried out by applying a thin
  layer
• Aqueous protein solution is immobilized in a
  solid hydrophilic support.
• Solid matrix with pores which are used
• paper
• starch
• cellulose acetate
• polyacrylamide
• agar/agarose
• Molecules in the sample move through porous
  matrix at different velocity.

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• Electrophoresis can be one dimensional (i.e.
  one plane of separation) or two dimensional.
• One dimensional electrophoresis is used for
  most routine protein and nucleic acid
  separations. Two dimensional separation of
  proteins is used for finger printing , and when
  properly constructed can be extremely accurate in
  resolving all of the proteins present within a
  cell (greater than 1,500).
• Most common stabilizing media are
  polyacrylamide or agarose gels.

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Buffers

• Function of buffer
• 1. carries the applied current
• 2. established the pH
• 3. determine the electric charge on the solute
• High ionic strength of buffer
• produce sharper band
• produce more heat
• Commonly used buffer
• Barbital buffer Tris-EDTA for protein
• Tris-acetate-EDTA Tris-borate-EDTA (50mmol/L
  pH 7.5-7.8)

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• Zone electrophoresis
• Much simple method
• Much greater resolution
• Require small sample
• Acetate cellulose support medium
• Protein separation depends on
• Type and number of ionizable side chains of
  amino acids - R.
• Size of net charge (positive or negative).
• Negatively charged proteins move towards the
  anode.
• Positively charged proteins move towards the
  cathode.

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Stripe of cellulose acetate
Electrophoresis
Major protein components separate into discrete
zones
Densitometer tracing density of zones is
proportional to the amount of protein
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Example of application of zone electrophoresis in
clinical practice
Hypergamaglobulinemia Hypogamaglobulinemia
Normal serum
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Gel electrophoresis

• Gel is a colloid in a solid form (99 is
  water).
• Gel material acts as a "molecular sieve.
• During electrophoresis, macromolecules are
  forced to move through the pores when the
  electrical current is applied.

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Support media

• Agarose and polyacrylamide gels are
  across-linked, spongelike structure
• It is important that the support media is
  electrically neutral. Presence of charge group
  may cause
• -Migration retardation
• -The flow of water toward one or the other
  electrode so called Electroendosmosis (EEO),
  which decrease resolution of the separation

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Agarose highly purified polysaccharide derived
from agar (extracted from seeweed), long sugar
polymers held together by hydrogen and
hydrophobic bonds. Acrylamide (CH2CH-CO-NH2) Pol
yacrylamide gel structure held together by
covalent cross-links
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Agarose gels

• For the separation of (1) large protein or
  protein complex (2) polynucleotide 50-30,000
  base-pairs
• The pore size is determined by adjusting the
  concentration of agarose in a gel (normally in
  the rank of 0.4-4

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Polyacrylamide gels
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• SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
• SDS (also called lauryl sulfate) - anionic
  detergent
• Molecules in solution with SDS have a net
  negative charge within a wide pH range.
• A polypeptide chain binds amounts of SDS in
  proportion to its relative molecular mass.
• The negative charges on SDS destroy most of the
  complex structure of proteins, and are strongly
  attracted toward an anode (positively-charged
  electrode) in an electric field.

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Diagrams of vertical slab gel assembly
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Determination of molecular mass
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Commonly used protein stains
Stain Detection limit Ponceau S 1-2
mg Amido Black 1-2 mg Coomassie
Blue 1.5 mg India Ink 100
ng Silver stain 10 ng Colloidal
gold 3 ng
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Staining with Coomasie blue
1 2 3
1 2 3
Assesment of individual lines
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An ethidium-stained gel photographed under UV
light
Each band that you see is a collection of
millions of DNA molecules, all of the same
length!!
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Western blott technique

• Western blot (also called immunoblot) is a
  technique to detect specifically one protein in a
  mixture of large number of proteins and to obtain
  information about the size and relative amounts
  of the protein present in different samples.
• In first proteins are separated using
  SDS-polyacrylamide gel electrophoresis.
• Then they are moved onto a nitrocellulose
  membrane. The proteins retain the same pattern of
  separation they had on the gel.

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• An antibody is then added to the solution which
  is able to bind to its specific protein and forms
  an antibody-protein complex with the protein of
  interest. (In fact there is no room on the
  membrane for the antibody to attach other than on
  the binding sites of the specific target
  protein).
• Finally the nitrocellulose membrane is
  incubated with a secondary antibody, which is an
  antibody-enzyme conjugate that is directed
  against the primary antibody.
• The location of the antibody is revealed by
  incubating it with a substrate that the attached
  enzyme converts to a product that can be seen and
  followed and then photographed.

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Isoelectric focusation
Proteins are separated in pH gradient. Protein
migrate into the point where its net charge is
zero isoelectric pH. Protein is positively
charged in solutions at pH values below its
pI. Protein is negatively charged in solution at
pH above its pI.
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Two-dimensional gel electrophoresis (2-D
electrophoresis )

• In the first dimension, proteins are resolved
  in according to their isoelectric points (pIs)
  using immobilized pH gradient electrophoresis
  (IPGE), isoelectric focusing (IEF), or
  non-equilibrium pH gradient electrophoresis.
• In the second dimension, proteins are separated
  according to their approximate molecular weight
  using sodium dodecyl sulfate poly-acrylamide-elect
  rophoresis (SDS-PAGE).

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Electrophoreogram of the mixture of proteins
Protein maps are compare with control pattern
of normal healthy person and abnormalities are
analysed
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Capillary electrophoresis Capillaries are
typically of 50 µm inner diameter and 0.5 to 1 m
in length. Due to electroosmotic flow, all
sample components migrate towards the negative
electrode. The capillary can also be filled with
a gel, which eliminates the electroosmotic flow.
Separation is accomplished as in conventional gel
electrophoresis but the capillary allows higher
resolution, greater sensitivity, and on-line
detection.
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Electroosmotic flow The surface of the silicate
glass capillary contains negatively-charged
functional groups that attract positively-charged
counterions. The positively-charged ions migrate
towards the negative electrode and carry solvent
molecules in the same direction. This overall
solvent movement is called electroosmotic flow.
During a separation, uncharged molecules move at
the same velocity as the electroosmotic flow
(with very little separation). Positively-charged
ions move faster and negatively-charged ions move
slower.