Vous êtes sur la page 1sur 37

12/11/2008

Dr. LOO HIAN DAO


CHAIRMAN of DEPARTEMENT BIOCHEMISTRY
SCHOOL of MEDICINE WIJAYA KUSUMA UNIVERSITY
SURABAYA

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.

12/11/2008

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.

A method of separating substances,


especially proteins, and analyzing
molecular structure based on the rate of
movement of each component in a
colloidal suspension while under the
influence of an electric field.

12/11/2008

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.

12/11/2008

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.

12/11/2008

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

In the case of nucleic acids, the direction of migration, from


negative to positive electrodes, is due to the naturallyoccurring negative charge carried by their sugar-phosphate
backbone.
Double-stranded DNA fragments naturally behave as long
rods, so their migration through the gel is relative to their
radius of gyration, or, for non-cyclic fragments, size. Singlestranded DNA or RNA tend to fold up into molecules with
complex shapes and migrate through the gel in a
complicated manner based on their tertiary structure.
Therefore, agents that disrupt the hydrogen bonds, such as
sodium hydroxide or formamide, are used to denature the
nucleic acids and cause them to behave as long rods again.
Gel electrophoresis of large DNA or RNA is usually done by
agarose gel electrophoresis.

12/11/2008

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.

12/11/2008

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.

AGAROSE GEL ELECTROPHORESIS

Agarose gel electrophoresis is a method used in


biochemistry and molecular biology to separate
DNA, RNA, or protein molecules by size. This
is achieved by moving negatively charged
nucleic acid molecules through an agarose
matrix with an electric field (electrophoresis).
Shorter molecules move faster and migrate
further than longer ones.

12/11/2008

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.

AGAROSE GEL ELECTROPHORESIS

Agarose gel electrophoresis is a method


for separating and visualizing
DNA fragments produced by restriction
digestion of DNA. The fragments are
separated by charge and size by
forcing them to move through a agarose
gel matrix which is subjected to an
electric field. The electric field is
generated by applying potential
(voltage) across an electrolytic solution
(buffer). Agarose is a marine
colloid purified from algae. When
boiled in an aqueous buffer it dissolves,
then upon cooling solidifies to a gel. It
can be molded into a slab with wells for
DNA samples just like jello can be
molded into many fun shapes. The
pouring of a gel and the set-up of a
submarine gel electrophoresis tank is
diagramed below. Your instructor will
show you the equipment in our lab.

12/11/2008

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.

AGAROSE GEL ELECTROPHORESIS


Factors affecting migration
The most important factor is the length of the DNA molecule, smaller
molecules travel farther. But conformation of the DNA molecule is also
a factor. To avoid this problem linear molecules are usually separated,
usually DNA fragments from a restriction digest, linear DNA PCR
products, or RNAs.
Increasing the agarose concentration of a gel reduces the migration
speed and enables separation of smaller DNA molecules. The higher
the voltage, the faster the DNA migrates. But voltage is limited by the
fact that it heats and ultimately causes the gel to melt.
Conformations of a DNA plasmid that has not been cut with a
restriction enzyme will move with different speeds (slowest to fastest):
nicked or open circular, linearised, or supercoiled plasmid.

12/11/2008

AGAROSE GEL
ELECTROPHORESIS

Visualisation: EtBr and dyes

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 GEL ELECTROPHORESIS


Resolution limits
Gel electrophoresis can be used for the separation of DNA fragments ranging from
50 base pair to several megabases (millions of bases). However, it is normally used
in a range of 100 bp to 20 kbp. Typical run times are about an hour.
Small nucleic acids are better separated by polyacrylamide gels, large DNA molecules
are only able to move end-on in a process called "reptation" and are more difficult to
separate. In general higher concentrations of agarose are better for larger
molecules; it will exaggerate the distances between bands. The disadvantage of
higher concentrations is the long run times (sometimes days). Instead these gels
should be run with a pulsed field electrophoresis (PFE), or field inversion
electrophoresis.

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.

10

12/11/2008

MEDIA FOR ELECTROPHORESIS

Buffers

AGAROSE GEL
ELECTROPHORESIS

There are a number of buffers used for


agarose electrophoresis. The most common
being: tris acetate EDTA (TAE),
Tris/Borate/EDTA (TBE) and Sodium
borate(SB). TAE has the lowest buffering
capacity but provides the best resolution
for larger DNA. This means a lower voltage
and more time, but a better product. SB is
relatively new and is ineffective in resolving
fragments larger than 5 kbp; However, with
its low conductivity, a much higher voltage
could be used (up to 35 V/cm), which means
a shorter analysis time for routine
electrophoresis. As low as one base pair size
difference could be resolved in 3% agarose
gel with an extremely low conductivity
medium (1 mM Lithium borate

11

12/11/2008

AGAROSE GEL ELECTROPHORESIS


Analysis
After electrophoresis the gel is illuminated
with an ultraviolet lamp (usually by placing it
on a light box, while using protective gear to
limit exposure to ultraviolet radiation) to view
the DNA bands. The ethidium bromide
fluoresces reddish-orange in the presence of
DNA. The DNA band can also be cut out of
the gel, and can then be dissolved to retrieve
the purified DNA. The gel can then be
photographed usually with a digital or
polaroid camera. Although the stained nucleic
acid fluoresces reddish-orange, images are
usually shown in black and white

12

12/11/2008

PREPARING AGAR

13

12/11/2008

BOILING AGAR

Preparing an Agarose Gel


DNA gels are made of agarose, a highly purified
agar, which is heated and dissolved in a buffer
solution. The agarose molecules form a matrix
with pores between them. The more
concentrated the agarose, the smaller the
pores. We will be using a 0.8% agarose gel
(there are 0.8 grams of agarose per 100 mL of
buffer) because we are looking at large DNA
fragments thousands of base pairs in length. A
2% agarose gel separates DNA fragments which
differ in length by as few as 50-100 base pairs.
Before beginning, double check the size of the
gel you are preparing. All volumes and weights
are given for a 50 mL gel.

14

12/11/2008

PREPARING TRAY

TAPED TRAY

15

12/11/2008

COOLING AGAR

ADD ETHIUM BROMIDE

16

12/11/2008

FINISHED TRAY

POUR AGAR INTO TRAY

17

12/11/2008

POUR AGAR INTO TRAY

INSERT COMB

18

12/11/2008

MAKE THE WELLS

ADDING BUFFER

19

12/11/2008

ADD RUNNING BUFFER

SAMPLE LOADED

20

12/11/2008

ELECTROPHORESIS SET UP

START POWER SUPPLY

21

12/11/2008

ELECTROPHORESIS RUNNING

RUNNING ELCTROPHORESIS

22

12/11/2008

ELECTROPHORESIS POWER SUPPLY

ELECTROPHORESIS

23

12/11/2008

MICROPIPET

Staining the DNA


The DNA must be stained in order to be seen. DNA
can be stained with fluorescent or chemical dyes.
Research laboratories use ethidium bromide, an
ultraviolet (UV) fluorescent stain, because it shows
very small amounts of DNA and is faster to use.
Ethidium bromide, however, can cause cancer and
mutate DNA. We will use methylene blue, a chemical
dye, which binds to DNA. Methylene blue may stain
your hands and clothes if you spill it, but it is not
toxic.
Hint: Wear gloves when working with methylene
blue.

24

12/11/2008

Staining the DNA

Native Gel Electrophoresis


Native Gel Electrophoresis is a technique used mainly in protein electrophoresis
where the proteins are not denatured and therefore separated based on their
charge-to-mass ratio.
The two main types of native gels used in protein electrophoresis are
polyacrlylamide gels and agarose gels.
Polyacrylamide gel electrophoresis (PAGE) is used for proteins ranging in size from
5 to 2,000 kDa due to the uniform pore size provided by the polyacrylamide gel.
Pore size is controlled by controlling the concentrations of acrylamide and bisacrylamide powder used in creating a gel. Care must be used when creating this
type of gel, as acrylamide is a potent neurotoxin in its powdered form. The other
type of gel used is agarose gel. Agarose gels are the most commonly used gels for
electrophoresis. They do not have a uniform pore size, but are optimal for
electrophoresis of proteins that are larger than 200 kDa.
Unlike SDS-PAGE type electrophoreses, Native gel electrophoresis does not use a
charged denaturing agent. The molecules being separated (usually proteins)
therefore differ in Molecular mass and intrinsic charge and experience different
electrophoretic forces dependant on the ration of the two. Since the proteins
remain in the native state they may be visualised not only by general protein
staining reagents but also by specific enzyme-linked staining.

25

12/11/2008

SEPARATION PROTEIN

AGAROSE GEL ELECTROPHORESIS

26

12/11/2008

AGAROSE GEL ELCTROPHORESIS

AGAROSE GEL ELECTROPHORESIS

27

12/11/2008

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.

28

12/11/2008

AGAROSE GEL ELECTROPHORESIS

Visualization of enzyme restriction

29

12/11/2008

Serum Protein Electrophoresis--Normal Pattern

Doctor diagnose Salmonella typhi


Pulsed-field gel
electrophoresis analysis of
isolates from our patient
(lanes 2, 3) and from
patients from several
California counties (lanes 4 7, 9 - 14), demonstrating
that the 2 samples taken
from the patient in Texas
(lane 2) and in California
(lane 3) represented the
same clone, which differed
from the California samples

30

12/11/2008

DNA PURIFICATION

FORENSIC DIAGNOSTIC

31

12/11/2008

BRIDGE
ELECTROPHORESIS

HORIZONTAL ELECTROPHORESIS

32

12/11/2008

Sodium dodecyl sulphate polyacrylamide gel


electrophoresis (SDS-PAGE)
This is a very common method of
gel electrophoresis for separating
proteins by mass. SDS-PAGE was
first employed by U.K Laemmli and
known as Laemmli method. The
proteins are dissolved in sodium
dodecyl sulfate (SDS), a detergent
that breaks up the interactions
between proteins, and then
electrophorised. The smallest
molecules move through the gel
faster, while larger molecules take
longer and result in bands closer to
the top of the gel.

Difference gel electrophoresis


Difference gel electrophoresis (DIGE) is
a form of gel electrophoresis where up
to three different protein samples can
be labeled with fluorescent dyes (for
example Cy3, Cy5, Cy2) prior to twodimensional electrophoresis. After the
gel electrophoresis, the gel is scanned
with the excitation wavelength of each
dye one after the other. This technique
is used to see changes in protein
abundance (for example, between a
sample of a healthy person and a
sample of a diseased person).

33

12/11/2008

VERTICAL ELECTROPHORESIS

The CSL omniPAGE range of Vertical Gel Units


combines ease of use with high resolution
separations. Two sizes, Mini 10 x 10cm and Maxi 20 x
20cm, share a host of common features including a
guaranteed leak proof seal required for trouble free,
rapid and uncomplicated gel casting. Utilising a built
in gel running module eliminates time consuming
transfer of glass plates during casting, a process
which can cause gel damage and
misalignment within the glass plates. Glass plates
with permanently bonded spacers guarantee perfect
spacer alignment. The glass plate sandwich is simply
inserted between pressure bars and a minimum of
screws tightened only one screw per side for
omniPAGE Mini. This guarantees fast set up times
while even pressure bars and ultra soft seals
guarantee leak proof casting. Once the gel has
polymerized, the gel running module is simply
inserted into the gel tank for 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.

34

12/11/2008

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.

35

12/11/2008

PULSE FIELD GEL ELECTROPHORESIS

Pulse field gel electrophoresis


(PFGE) of Blnl enzyme from 10
strains of Shigella flexneri

The theory behind why PFGE


works pertains to the mobility of
larger DNA fragments. While in
general small fragments can
wind their way through the gel
matrix more easily than large
DNA fragments, a threshold
length exists where all large
fragments will run at the same
rate. But with a continuous
changing of directions every few
seconds or fraction of a second,
the various lengths of DNA react
to the change at differing rates.
That is, larger pieces of DNA will
be slower to begin moving in the
opposite direction while smaller
pieces will be quicker to change
direction. Over the course of
time with the consistent
changing of directions, each
band will begin to separate
more and more even at very
large lengths. Thus separation of
very large DNA pieces using
PFGE is possible.

2-dimensional gel electrophoresis

36

12/11/2008

Virtual Laboratory Gel electrophoresis

37