Transcript Isoelectric focusing

ELECTROPHORESIS
Prof/ Azza abd al baky
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Electrophoresis is the migration of charged
molecules,particles or ion in a liquid medium under
the influence of an electric field
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Various types – defined by support used
1.
Paper – amino acids, small peptides
2.
Polyacrylamide – Proteins, small DNA/RNA (<500bp)
3.
Agarose – DNA/RNA
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Good preparative and analytical method
Proteins
move in the
electric field. Their relative
speed depends on the
charge, size, and shape of
the protein
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.
General Procedure
Factors
affecting
migration rate
Sample
Electric field
Buffer
Supporting
media
The sample
Charge
Size
Shape
The electric field
Current
Voltage
Resistance
Heat
The Buffer
Composition
Concentration
PH
The Supporting media
Adsorption
Electro-osmosis
Molecular sieving
Types of ELECTROPHORESIS
Zone electrophoresis
Slab Gel Electrophoresis
Disc electrophoresis
lsoelectric Focusing electrophoresis
Two-Dimensional (2D) Electrophoresis
Capillary electrophorsis
Microship electrophorsis
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 uses a cross-linked polymers
(agarose) that contain various pores.
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Pores allow molecular sieving, where molecules e.g.
DNA, can be separated based upon there mobility
through the gel.
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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 40°C.
It is fragile because of the formation of weak
hydrogen bonds and hydrophobic bonds.
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Used to separate most proteins and small
oligonucleotides because of the presence of small
pores.
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1.
2.
3.
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1.
2.
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
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!
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

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)
IEF
4-6.5 pH
gradient
Zavialov
A.
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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1.
2.
3.
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 – 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.
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)
1.
Separation of proteins using SDS PAGE
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2. Transfer of the proteins onto e.g. a
nitrocellulose membrane (blotting)
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3. Immune reactions
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4. Visualization
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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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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
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
A.bent applicators.
B.incorporation of an air bubble during sample
application.
C.over application of sample.
D.excessive drying of the electrophoretic support
before or during electrophoresis.
4.
5.
6.
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.
‫الحمد هلل رب‬
‫العالمين‬
‫‪‬‬