Transcript Electrophoresis - MITCON Biopharma

Electrophoresis
Electrophoresis
• Separation
technique based on the movement of
analyte through a conductive medium in response
to an applied electrical field.
• The medium is usually a buffered aqueous
solution.
• In the absence of other factors, cationic species
will migrate towards the cathode and anionic
species towards the anode.
Theory of Electrophoresis
 Electrophoretic separations are based upon the fact that the electrical
force (F) on a charged particle (ion) in an electrical field (E) is
proportional to the charge of the particle (q),
F = qE
 The migration of the charged particle in the electric field, called the
electrophoretic mobility (μ),
μ = v/E = q/f
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Dr Gihan Gawish
Theory of Electrophoresis
 Move at quite different depending on
 physical characteristics of the molecule
 i.e its molecular size.
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TYPES OF ELECTROPHORESIS
Free-Solution method/ capillary eclectrophoresis.
! Absence of a supporting/stabilizing medium.
! Sample is introduced into a tube filled with a
buffering liquid.
! A field is applied and species migrate based on
their charge to mass ratios.
! Tiselius - 1948 Nobel prize for his development of this approach for the
purification of proteins.
Most popular method is capillary electrophoresis
1- Moving Boundary Electrophoresis
 Electrophoresis in a free solution.
 the separation of colloids through electrophoresis
 the motion of charged particles through a stationary liquid
under the influence of an electric field.
Developed by Arne Tiselius in 1937. Tiselius was awarded the 1948 Nobel
Prize in chemistry for his work
Stabilizing media methods
•Presence of a supporting medium like paper,
•packing, or gel.
•Resemble chromatographic methods except that
•migration is based on an electrical field instead of
•a mobile phase.
•A number of methods have been based on this approach
including
•Electrochromatography
•Zone electrophoresis
•Electromigration
•Ionophoresis
2- Zone Electrophoresis
A drop of sample is applied in a band to a thin sheet of supporting
material, like paper, that has been soaked in a slightly-alkaline salt
solution .
Paper electrophoresis employs filter paper strips soaked in buffer
solution, usually diethylbarbituric acid and barbituric acid dissolved
in alkali (Veronal buffer), pH 8.6. A small volume of serum is placed
on the paper and a direct current passed for several hours

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3- Gel Electrophoresis
 The term "gel" in this instance refers to the matrix used
to contain, then separate the target molecules.
 In most cases the gel is a cross linked polymer whose
composition and porosity is chosen based on:
 the specific weight
 composition of the target to be analyzed.
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Introduction
Principles of nucleic acid separation by agarose gel
electrophoresis
Agarose gel electrophoresis is a routinely used method for separating
proteins, DNA or RNA. Nucleic acid molecules are size separated by
the aid of an electric field where negatively charged molecules migrate
toward anode (positive) pole. The migration flow is determined solely
by the molecular weight where small weight molecules migrate faster
than larger ones. In addition to size separation, nucleic acid
fractionation using agarose gel electrophoresis can be an initial step for
further purification of a band of interest. Extension of the technique
includes excising the desired “band” from a stained gel viewed with a
UV transilluminator
Gel Electrophoresis
larger nucleic acids
(greater than a few
hundred bases)
the preferred
matrix
*agarose.
 *Acrylamide, in contrast to *Polyacrylamide, is a
neurotoxin and must be handled using appropriate safety
precautions to avoid poisoning.
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Visualization
Ethidium bromide is the common dye for nucleic acid visualization. The early
protocol that describes the usage of Ethidium bromide (2,7-diamino-10-ethyl-9phenylphenanthridiniumbromide-) for staining DNA and RNA in agarose gels dates
as far back as 1970s.
Although with a lower efficiency compare to the double- stranded DNA, EtBr is
also used to stain single- stranded DNA or RNA.
Under UV illumination, the maximum excitation and fluorescence emission of EtBr
can be obtained from 500- 590 nm. Exposing DNA to UV fluorescence should be
performed rapidly because nucleic acids degrade by long exposures and thus, the
sharpness of the bands would be negatively affected.
Gel Electrophoresis- Visualization
Ethedium bromide
Silver
stained
Photograph
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Autoradiogram
Dr Gihan Gawish
Gel Electrophoresis- Visualization
 The bands observed (unknown molecular weight) can be compared to
those of the known (Molecular weight size markers) in order to
determine their size.
 Molecular weight size markers contain a mixture of molecules of
known sizes.
 marker run on one lane in the gel parallel to the unknown samples
Bands in different
lanes that end up
at the same
distance from the
top
contain molecules that
passed through the gel
with the same speed
means they are
approximately the
same size
An alternative dsDNA stain is SYBR Green I. Despite the fact that
SYBR Green is more expensive, it is 25 times more sensitive than
ethidium bromide
Since EtBr stained DNA is not visible in natural light, negatively
charged loading buffers are commonly added to DNA prior to
loading to the gel.
Loading buffers are particularly useful because they are visible in
natural light and they co-sediment with DNA.
Xylene cyanol and Bromophenol blue are the two common
dyes used as loading buffers and they run about the same speed as
DNA fragments that are 5000 bp and 300 bp respectively. The
other less frequently used progress markers are Cresol Red and
Orange G which run at about 125 bp and 50 bp, respectively
Agarose Gel
Stained with ethidium bromide (EtBR) to Visualize the DNA
slots where
DNA is loaded
1000 bp
700 bp
600 bp
500 bp
Screening PCR
products to test
for the presence
of specific DNA
sequences
molecular
weight
markers
correct
PCR
product
molecular
weight
markers
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Dr.Saba Abdi
Preparing and running standard agarose DNA gels
Several electrophoresis buffers can be used for fractionating nucleic acid such as,
Trisacetate- EDTA (TAE)
Tris-borate-EDTA (TBE)
For gel preparation,
Agarose powder electrophoresis grade is mixed with electrophoresis buffer to the
desired concentrations (usually with a range of 0,5-2%) then heated in a microwave
oven until completely dissolved. Ethidium bromide is usually added to the gel for
nucleic acid visualization. The mixture is cooled to 60 C and poured into the casting
tray for solidification.
Wells should be placed towards the negative electrode?
At the same time, ethidium bromide migrates in the reverse direction, meets and
couples with DNA fragments. DNA fragments are visualized by staining with ethidium
bromide when adequate migration has occurred. Then, this fluorescent dye intercalates
between bases of DNA and RNA
Factors listed below are effecting the mobility of DNA fragments
in agarose gels
Agarose concentration
In the gel, the distance between DNA bands of a given length is determined
by the percent agarose. Higher concentrations have the disadvantage of long
run times.
Most agarose gels are prepared with the agarose concentrations ranging 0.7%
(good separation or resolution of large 5–10kb DNA fragments) to 2% (good
resolution for small
0.2–1kb fragments)
Voltage
Migration of fragments in an agarose gel depends on the difference in electric current.
Different optimal voltages are required for different fragment sizes. For instance, the
best resolution for fragments larger than 2 kb could be obtained by applying no more
than 5 volts per cm to the gel
Electrophoresis buffer
Various buffers are used for agarose electrophoresis. The two most common buffers
for nucleic acids are Tris/Acetate/EDTA (TAE) and Tris/Borate/EDTA (TBE). DNA
fragments migrate with different rates in these two buffers due to differences in ionic
strength.
Buffers not only establish an ideal pH, but provide ions to support conductivity. In
general, the ideal buffer should produce less heat, have a long life and a good
conductivity.
For example, deviations from the optimal concentration of the buffer (over
concentrated) could produce enough heat to melt the gel
Applications of Gel Electrophoresis
Forensics,
 The
results
can
be
analyzed
Molecular biology,
quantitatively by visualizing the gel
Genetics
with UV light and a gel imaging
 Microbiology
device.
 Biochemistry.
 The
image is recorded with a
computer operated camera