Introduction To Biotech Instrumentation: Electrophoresis I

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Introduction To Biotech Instrumentation
Electrophoresis (I)

• Yao-Te Huang
• Oct 22, 2009

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Electrophoresis (1) The movement of charged
particles under the influence of an electric
field is called electrophoresis
- - - -

q
FeE q
Fdfv
E the electric field

• E ? q f ? v
• where Ethe electric field
• qthe net charge on the molecule
• fthe frictional coefficient (a
  function
• of the size shape of molecule)
• vthe velocity of the molecule

The migration of a sample during electrophoresis
is a function of its size, shape, and charge, in
general!
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Electrophoresis (2)

• Mobility (?)?v/E (defined as the velocity per
  unit of electric field), so ?q/f.
• In practice, relative mobility
• mobility of sample divided by mobility of
  standard
• (distance of sample migration)/(distance of
  standard migration)

In practice, we use a tracking dye as a reference
standard
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Zonal electrophoresis

• Zonal electrophoresis in which, a thin layer or
  zone of the macromolecule solution is
  electrophoresed through some kind of matrix

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Ferguson plot Log(µ) vs. C
The gel acts like a molecular sieve. A
macromolecule can not move easily through a
network as it can when free in solution.

• Log(µ) log(µ0)-kC
• where µis the (relativity) mobility at the gel
  concentration C
• µ0 is the (relativity) mobility when C0
• k is a constant which depends on MW of the
  sample

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Ferguson plot (2)
Line 1 Some are small, but highly charged , and
will have a large mobility but a small k. Line
2 they may be large and highly charged. Line
3 they are small, and have a small charge.
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Gel Electrophoresis of Proteins

• Outline
• 1-dimensional (1-D) polyacrylamide gel
  electrophoresis (1D PAGE)
• IEF (isoelectric focusing)
• 2-dimensional (2-D) gel electrophoresis
• Capillary electrophoresis

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1-D PAGE (PolyAcrylamide Gel Electrophoresis )
Different samples are loaded in wells or
depressions at the top of the polyacrylamide
gel. The proteins move into the gel when an
electric field is applied. The gel Minimizes
convection currents caused by small temperature
gradients, as well as protein movements other
than those induced by the electric field.
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Polyacylamide gel electrophoresis

• The migration of molecules through polyacrylamide
  gel is largely affected by the structure of the
  gel (the pore size of the gel).
• The separation of molecules through gel is based
  on both molecular sieving and electrophoretic
  mobility

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The compounds used to construct the polymer gel
matrix are
(1)
Monomer of the polymer
(3)
Ammonium persulfate (NH4)2S2O8
Catalysts of gel formation
(2)
Cross-linking agent
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When ammonium persulfate is dissolved in water,
it produces free radicals.
A linear polymer is not enough! Needs
cross-linking them to one another
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Doing polymerization in this way produces a net
of acrylamide chains. Two parameters determine
the pore size (a) the amount of acrylamide used
per unit volume of reaction medium (b) the
degree of cross-linkage
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An approximation of the pore sizes produced from
various concentrations (w/v) of acrylamide
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The average pore size reaches a minimum when 5
of the total acrylamide used is
N,N-methylene-bis(acrylamide)
Thus, in many applications, the concentration of
bis(acrylamide) is fixed at 5 of the total
acrylamide.
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Discontinuous gel electrophoresis
The purpose of using stacking gel eliminating
the sample volume effect. A very high localized
voltage gradient occurs between the chloride ions
and the glycinate ions, resulting in the
concentration of the protein sample in a tight
disc between glycinate and chloride ions
pI of Gly 5.97 Gly moves much slower at
pH6.8 than pH8.8
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The stacking gel having lower percentage of
acrylamide has large pore size The resolving
gel having higher percentage of acrylamide has
smaller pore size
-

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Electrophoresis unit
A Teflon comb to generate wells for sample
loading Analytic 10-16 wells Preparative 1-2
wells
Spacers inserted between two glass plates to
allow the gel slab to be made
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SDS-PAGE
SDS sodium dodecyl sulfate (CH3-(CH2)11-SO4-Na)
The action of SDS (1) to denature protein (2) to
coat protein with a uniform negative charge
(about one SDS molecule per two amino acid
residues) and constant charge-to-mass ratio
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SDS-PAGE composition of SDS-PAGE sample buffer

• SDS
• ?-mercaptoethanol (HSCH2CH2OH) to reduce
  disulfide bonds of proteins
• Glycerol (making sample denser, convenient for
  sample loading)
• Bromophenol blue a tracking dye
• Tris (pH6.8)
• Heated at 100 C for several minutes
• when mixed with a PAGE protein sample

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Staining the gel
Coomassie blue (R250 or G250) staining
The dyes binds to proteins via non-covalent
interactions (mainly ionic interactions,
although van der Waals interactions are probably
involved) About 0.1ug-of-protein sensitivity
Staining solution Coomassie blue, MeOH, acetic
acid, and water De-staining solution MeOH,
acetic acid, and water
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Silver staining
About ng-of protein sensitivity

• Silver nitrate is added under the acidic
  condition
• gtSilver ions bind to protein (basic groups)
• gtformaldehyde is added under basic condition
• gtsilver ion is reduced to metallic silver
• gtNucleation of the growth of the globular
• metallic silver grains proceeds

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The use of SDS-PAGE to estimate the MW of proteins
The mobilities of proteins are a linear function
of the logarithms of their molecular weights
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Estimating the molecular weight of a protein.
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Polyacrylmaide gel with lower percentage used
for separating proteins with higher
MWs Polyacrylamide gel with higher percentage
used for separating proteins with lower MWs
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IEF (IsoElectric Focusing) a procedure used to
determine the pI of a protein or to separate
proteins based on their pI values
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The pI values of some proteins
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2-dimensional gel electrophoresis permits high
resolution of a complex mixture of proteins
The first dimension IEF The second dimension
SDS-PAGE
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Proteins from E. coli were separated by
two-dimensional gel electrophoresis, resolving
more than a thousand different proteins
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Capillary Electrophoresis (1)

• Separation occurs in a fused quartz capillary,
  typically 50-100 µm in a diameter and 20-60 cm
  long.
• The high voltage is applied, and allows for rapid
  separation ? often in minutes.
• Detection is usually by UV absorbance or
  fluorescence as components pass through a given
  point in the capillary.
• Advantage extremely small amount of sample is
  required (femtomole (10-15 mol) by UV detection
  or zeptomole (10-21 mol) by fluorescence)
• Disadvantage some proteins may be absorbed or
  denatured by interaction with the surface. Such
  an effect can be reduced by coating the capillary
  with a polymeric material.

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Capillary Electrophoresis (2)
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Capillary Electrophoresis (contd.)
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Principles of Capillary electrophoresis
Electroosmotic flow the positively charged
Na/H ions are electrophoretically attracted
toward the cathode and carry the hydration layer
of water (which is H-bonded to bulk water) with
them Electrophoretic flow positively charge ions
are attracted to the cathode negatively charged
ions are attracted to the anode. Thus, during
capillary electrophoresis, the precise mobility
of an individual ion will be the combination of
these two flows. Positively charged ions moving
toward the cathode Uncharged molecules moving
toward the cathode Negatively charged ions
depending upon the relative strength of the two
flows
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Interactions between two flows
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Apparatus used for Capillary Electrophoresis
The voltage is high lethal!
Capillaries have small internal volumes and large
surface areas!
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Western blotting

• A protein mixture is electrophoresed
• through an SDS gel, and then
• transferred from the gel onto a
• membrane. (b) The membrane is flooded
• with a solution of primary antibody
• (Ab1) specific for the desired protein
• . Only the band containing this protein
• binds to the antibody, forming a layer
• of antibody molecules (although their
• position can't be seen at this point).
• After sufficient time for binding, the
• membrane is washed to remove
• unbound Ab1. (c) In the development
• step, the membrane first is incubated
• with a secondary antibody (Ab2) that
• binds to the bound Ab1. This secondary
• antibody is covalently linked to alkaline
• phosphatase (or horseradish-peroxidase)
• , which catalyzes a chromogenic reaction.

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AP (Alkaline Phosphatase)
HRP (horseradish -peroxidase)
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Western blotting contd.
Proteins on an SDS-polyacrylamide gel are
transferred to a polymer sheet and stained with
radioactive antibody. A band corresponding to the
protein to which the antibody binds appears in
the autoradiogram