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Electrokinetic phenomena
Electrokinetic phenomena is a
family of several different effects
that occur in heterogeneous fluids
or in porous bodies filled with fluid.
The term heterogeneous here
means a fluid containing particles.
Particles can be solid, liquid or gas
bubbles with sizes on the scale of a
micrometer or nanometer.
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Electrokinetic phenomena
The complete family of electrokinetic phenomena includes:
Electrophoresis, as motion of particles under influence of electric field;
Electro-osmosis, as motion of liquid in porous body under influence of
electric field;
Diffusiphoresis, as motion of particles under influence of a chemical
potential gradient;
Capillary osmosis, as motion of liquid in porous body under influence of
the chemical potential gradient;
Sedimentation potential, as electric field generated by sedimenting
colloid particles;
Streaming potential/current, as either electric potential or current
generated by fluid moving through porous body, or relative to flat surface;
Colloid Vibration Current, as electric current generated by particles
moving in fluid under influence of ultrasound;
Electric Sonic Amplitude, as ultrasound generated by colloidal
particles in osccilating electric field.
Electrophoresis
It was discovered by Reuss in 1809. He
observed that clay particles dispersed in water
migrate under influence of an applied electric
field.
Generally, electrophoresis is the motion of
dispersed particles relative to a fluid under the
influence of an electric field that is space
uniform. Alternatively, similar motion in a
space non-uniform electric field is called
dielectrophoresis.
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Electrophoresis
Electrophoresis is a technique used to
separate and sometimes purify
macromolecules - especially proteins and
nucleic acids - that differ in size, charge or
conformation. As such, it is one of the most
widely-used techniques in biochemistry and
molecular biology.
When charged molecules are placed in an
electric field, they migrate toward either the
positive or negative pole according to their
charge. In contrast to proteins, which can
have either a net positive or net negative
charge, nucleic acids have a consistent
negative charge imparted by their phosphate
backbone, and migrate toward the anode.
Electrophoresis
Proteins and nucleic acids are electrophoresed within a
matrix or "gel". 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.
The gel itself is composed of either agarose or
polyacrylamide, each of which have attributes suitable to
particular tasks:
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: you simply mix agarose powder
with buffer solution, melt it by heating, and pour the gel. 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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Electrophoresis
Polyacrylamide is a cross-linked polymer of acrylamide. The length of the
polymer chains is dictated by the concentration of acrylamide used, which
is typically between 3.5 and 20%. Polyacrylamide gels are significantly
more annoying to prepare than agarose gels. Because oxygen inhibits the
polymerization process, they must be poured between glass plates (or
cylinders).
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.
Polyacrylamide gels have a rather small range of separation, but very
high resolving power. In the case of DNA, polyacrylamide is used for
separating fragments of less than about 500 bp. However, under
appropriate conditions, fragments of DNA differing is length by a single
base pair are easily resolved. In contrast to agarose, polyacrylamide gels
are used extensively for separating and characterizing mixtures of
proteins.
Electrophoresis Theory
The separation by electrophoresis depends on differences in the migration
velocity of ions or solutes through the given medium in the electric field.
The electrophoretic migration velocity (up) of an analyte is:
up = pE
Where E is the electric field strength and p is the electrophoretic
mobility.
The electrophoretic mobility is inversely proportional to frictional forces in
the buffer and directly proportional to sample's the ionic charge. The
forces of friction against an ion is dependent on size of the ion and the
viscosity () of the medium. Analytes with different frictional forces or
different charges will separate from one another when they move through
a buffer. At a given pH, the electrophoretic mobility of an analyte is:
Where r is the radius of the analyte and z is the net charge of the analyte.
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Electrophoresis Theory
The differences in the ratio of charge to size of the
analytes cause differences in electrophoretic
mobility. Small and highly charged analytes have
greater mobility, whereas large and low charged
analytes have lower mobility.
Electrophoretic mobility is an indication of speed of
a given analyte in a give medium. It is the balance of
electrical force that acts in favor and the frictional
force that acts against the motion. These two forces
remain steady during electrophoresis; therefore
electrophoresis mobility is a constant for a given ion
under a given set of conditions. Based on this
characteristic property of ion or solute, it can be
separated using electrophoresis.
Gel Electrophoresis
Nucleic acids
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Gel Electrophoresis
PROTEIN
Proteins, unlike nucleic acids, can have varying charges and complex
shapes, therefore they may not migrate into the gel at similar rates, or
at all, when placing a negative to positive EMF on the sample. Proteins
therefore, are usually denatured in the presence of a detergent such
as sodium dodecyl sulfate/sodium dodecyl phosphate (SDS/SDP) that
coats the proteins with a negative charge.Generally, the amount of
SDS bound is relative to the size of the protein (usually 1.4g SDS per
gram of protein), so that the resulting denatured proteins have an
overall negative charge, and all the proteins have a similar charge to
mass ratio. Since denatured proteins act like long rods instead of
having a complex tertiary shape, the rate at which the resulting SDS
coated proteins migrate in the gel is relative only to its size and not its
charge or shape.
Proteins are usually analyzed by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE), by native gel electrophoresis, by quantitative
preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE),
or by 2-D electrophoresis.
Applications of Electrophoresis
Electrophoresis has wide variety of applications in proteomics,
forensics, molecular biology, genetics, biochemistry, and
microbiology.
The common method of electrophoresis used in proteomics is
Polyacrylamide Gel Electrophoresis (PAGE). PAGE is used for
separation, identification, and purification of proteins. The proteins
can be analyzed for information about the mass, charge, and purity.
Different types of PAGE provide different types of information about
the proteins. SDS-PAGE is used for separating proteins based on their
molecular mass. SDS-PAGE is also used for protein identification and
quantitation, identification of disulfide bonds,determination of purity
of sample, and also blotting applications. Native-PAGE is used to
separate proteins in their native form without denaturing. QPNC-PAGE
is used to isolate active or native metalloproteins in biological
samples and to resolve properly and improperly folded metal
cofactor-containing proteins in complex protein mixtures. 2D-PAGE
separates modified proteins and provide information about various
modified states. 2D-PAGE and DIGE are used to study differential
expression of proteins in healthy and diseased tissues. Proteins that
are more abundant in disease tissue may be considered as diagnostic
disease markers or potential drug targets. Since a sinle 2-DE can
resolve thousands of proteins it is also used for cell map proteomics
as well as for characterizing subproteomes. Capillary electrophoresis
is employed for faster, efficient separation of proteins.
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Applications of
Electrophoresis
Electrophoresis is also used for separation and
analysis of DNA. Sequencing of DNA, analysis of
PCR products, separation and estimation of
the size of the DNA fragments from restriction
enzyme digestion are only some of the
applications. Gel electrophoresis is used in
DNA fingerprinting which has applications in
forensics. Certain DNA segments are
characteristic and vary from person to person.
These segments are cut at recognition sites by
restriction endonuclease enzymes and run on
gel by electrophoresis.The position and
number of bands on each lane of gel is
characterestic of the DNA sample and
considered as the genetic "fingerprint" of that
sample.
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AGAROSE GEL
ELECTROPHORESIS
Gel electrophoresis
apparatus - An agarose gel
is placed in this bufferfilled box and electrical
current is applied via the
power supply to the rear.
The negative terminal is at
the far end (black wire), so
DNA migrates toward the
camera.
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AGAROSE GEL
ELECTROPHORESIS
Applications
Estimation of the size of DNA molecules
following restriction enzyme digestion, e.g. in
restriction mapping of cloned DNA.
Analysis of PCR products, e.g. in molecular
genetic diagnosis or genetic fingerprinting
Separation of restricted genomic DNA prior to
Southern transfer, or of RNA prior to Northern
transfer.
The advantages are that the gel is easily
poured, does not denature the samples. The
samples can also be recovered.
The disadvantages are that gels can melt
during electrophoresis, the buffer can
become exhausted, and different forms of
genetic material may run in unpredictable
forms.
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AGAROSE GEL
ELECTROPHORESIS
The most common dye used for agarose gel electrophoresis is ethidium
bromide, usually abbreviated as EtBr. It fluoresces under UV light when
intercalated into DNA (or RNA). EtBr is a known carcinogen, however, and
alternatives are available.
SYBR Green I is another dsDNA stain, produced by Invitrogen. It is more
expensive, but 25 times more sensitive, and possibly safer than EtBr, though
there is no data addressing its mutagenicity or toxicity in humans.
SYBR Safe is a variant of SYBR Green that has been shown to have low enough
levels of mutagenicity and toxicity to be deemed nonhazardous waste under
U.S. Federal regulations.It has similar sensitivity levels to EtBr, but, like SYBR
Green, is significantly more expensive.
Loading buffers are added with the DNA in order to visualize it and sediment it
in the gel well. Negatively charged indicators keep track of the position of the
DNA. Xylene cyanol and Bromophenol blue are typically used. They run at
about 5000 bp and 300 bp respectively, but the precise position varies with
percentage of the gel.
Agarose
Agarose is purified from agar, a gelatinous substance isolated from seaweed or
human waste. Different purities of agarose are commercially available as are
agaroses with different melting properties. High purity low melt agarose is often
used if the DNA is to be extracted from the gel.
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Buffers
AGAROSE GEL
ELECTROPHORESIS
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PREPARING AGAR
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BOILING AGAR
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PREPARING TRAY
TAPED TRAY
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COOLING AGAR
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FINISHED TRAY
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INSERT COMB
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ADDING BUFFER
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SAMPLE LOADED
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ELECTROPHORESIS SET UP
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ELECTROPHORESIS RUNNING
RUNNING ELCTROPHORESIS
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ELECTROPHORESIS
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MICROPIPET
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SEPARATION PROTEIN
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GEL ELECTROPHORESIS
ELECTROPHORETOGRAM
An electrophoretogram is
the result of an
electrophoresis, which gives
the movement of charged
particles over time in a gel,
paper or another medium.
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DNA PURIFICATION
FORENSIC DIAGNOSTIC
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BRIDGE
ELECTROPHORESIS
HORIZONTAL ELECTROPHORESIS
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VERTICAL ELECTROPHORESIS
Capillary electrophoresis
Capillary electrophoresis (CE), also known as
capillary zone electrophoresis (CZE), can be used
to separate ionic species by their charge and
frictional forces. In traditional electrophoresis,
electrically charged analytes move in a
conductive liquid medium under the influence of
an electric field. Introduced in the 1960s, the
technique of capillary electrophoresis (CE) was
designed to separate species based on their size
to charge ratio in the interior of a small capillary
filled with an electrolyte.
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Capillary electrophoresis
The system's main components are a sample vial, source and destination vials, a capillary,
electrodes, a high-voltage power supply, a detector, and a data output and handling device.
The source vial, destination vial and capillary are filled with an electrolyte such as an
aqueous buffer solution. To introduce the sample, the capillary inlet is placed into a vial
containing the sample and then returned to the source vial (sample is introduced into the
capillary via capillary action, pressure, or siphoning). The migration of the analytes is then
initiated by an electric field that is applied between the source and destination vials and is
supplied to the electrodes by the high-voltage power supply. It is important to note that all
ions, positive or negative, are pulled through the capillary in the same direction by
electroosmotic flow, as will be explained. The analytes separate as they migrate due to their
electrophoretic mobility, as will be explained, and are detected near the outlet end of the
capillary. The output of the detector is sent to a data output and handling device such as an
integrator or computer. The data is then displayed as an electropherogram, which reports
detector response as a function of time. Separated chemical compounds appear as peaks
with different retention times in an electropherogram.
Capillary electrophoresis
The surface of the silicate
glass capillary contains
negatively-charged functional
groups that attract
positively-charged
counterions. The positivelycharged 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). Positivelycharged ions move faster and
negatively-charged ions move
slower.
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