Transcript Electrophoresis

ELECTROPHORESIS
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Electrophoresis refers to separation of compounds by
employing electrophoretic mobility i.e. movement of charged
molecules in response to an electric field.
Electrophoresis is carried out by adding the mixture of
compounds to a conductive medium followed by the application
of an electric field across the medium.
Positively charged molecules will migrate towards the negative
electrode while the negatively charged compounds will migrate
towards the positive electrode.
Neutral compounds will remain stationary. Proteins and
nucleic acids have different charge on them depending on the
medium pH.
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In certain cases a compound can be imparted
specific charge by using suitable chemicals e.g. ionic
detergents.
Depending on the medium in which an
electrophoretic separation is carried out
electrophoresis can be classified into two types:
•Gel electrophoresis
•Liquid phase electrophoresis
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Electrophoresis
• A separation technique often applied to the analysis of
biological or other polymeric samples
• Among the most powerful for estimating purity because of its
simplicity, speed, and high resolution, and also because there
is only a small probability that any of the components being
analyzed will be lost during the process of analysis
• Has frequent application to analysis of proteins and DNA
fragment mixtures and has been increasingly applied to the
analysis of nonbiological and nonaqueous sample
• The electric field doest not effect a molecule’s structure, and
it is highly sensitive to small difference in molecular charge,
size and sometimes shape
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Electrophoresis
Principles
• The fundamental principle behind electrophoresis is the existence of
charge separation between the surface of a particle and the fluid
immediately surrounding it
• An applied electric field acts on the resulting charge density, causing
the particle to migrate and the fluid around the particle to flow
• The electric fields exerts a force on the particle’s charge or surface
potential
• Two particles with different velocities will come to the rest in different
locations after a fixed time in an electric field The particle velocity is
related to the field strength by
(1) V: particle velocity, E: field strength or gradient (voltage per length),
U: apparent electrophoretic mobility.
• There are two contributions to this apparent electrophoretic mobility:
(2)
Uel: electrophoretic mobility of the charged particle
Uo: the contribution from electroosmotic flow.
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Modes of Electropheretic Separation
Gel electrophoresis
• In gel electrophoresis, migration takes place though a gel slab
• A common gel material for the study of proteins is cross-linked
polyacrylamide
• In most cases, the goal of experiment is to separate a sample
according to molar masses of its components
• However, the shape and charge will also determine the drift speed
• One way to avoid this problem and to effect separation by molar
mass is to denature the proteins in a controlled way
• Sodium dodecyl sulfate is an anionic detergent that is very useful in
this respect: it denatures proteins, whatever their initial shapes, into
rods by forming a complex with them
• Moreover, most proteins bind a constant amount of ion, so that the
net charge per protein is well regulated
• Under these conditions, different proteins in a mixture may be
separated according to size only
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Modes of Electropheretic Separation
Figure 1 SDS-PAGE (denaturing gel electrophoresis) and Western blot results for
bovine growth hormone (bGH) expressed as a C-terminal fusion to E. coli NusA
protein. (a) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
results with Coomassie blue staining. (b) Western blot results obtained by using rabbit
anti-bGH polyclonal antibody and visualized by means of chemiluminescence. Fusion
proteins were expressed at 37°C in E. coli by induction of the tac promoter. Equal
portions of cell lysate, soluble fraction, and insoluble fraction were loaded. Key: m,
markers; u, uninduced whole cell lysate; i, induced whole cell lysate; sol, soluble
fraction; ib, inclusion body fraction.
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Modes of Electropheretic Separation
Capillary electrophoresis
• The drift speeds attained by polymers in traditional electrophoresis
methods are rather low; as a result, several hours are often necessary
to effect good separation of complex mixtures
• One way to increase the drift speed is to increase the electric field
strength
• However, there are limits to this strategy because very large electric
fields can heat the large surfaces of an electrophoresis apparatus
unevenly, leading to nonuniform distribution of electrophoretic
mobilities and poor separation
• In capillary electrophoresis, the sample is diepersed in a medium
(such as methylcellulose) and held in a thin glass or plastic tube with
diameters ranging from 20 to 100 µm
• The small size of the apparatus makes it easy to dissipate heat when
large electric fields are applied
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Modes of Electropheretic Separation
Figure 2 Separation of proteins by open tubular capillary electrophoresis,
carried out in a 75 cm x 75 µm surface modified capillary at an applied voltage
of 75 kV. Peak identities: A, egg white lysozyme; B, horse heart cytochrome c;
C, bovine pancreatic ribonuclease a; D, bovine pancreatic α-chymotrypsinogen;
F, equinemyoglobin.
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Modes of Electropheretic Separation
Isoelectric focusing
• Naturally occurring macromolecules acquire a charge when dispersed in water
• An important feature of proteins and other biopolymers is that their overall charge
depends on the pH of the medium
• For instance, in acidic environments protons attach to basic groups and the net
charge is positive; in basic media the net charge is negative as a result of proton
loss
• At the isoelectric point, the pH is such that there is no net charge on the
biopolymer
• Consequently, the drift speed of the biopolymer depends on the pH of the
medium, with s = 0 at the isoelectric point
• Isoelectric focusing is an electrophoresis method that exploits the change of drift
speed with pH
• Consider a mixture of distinct proteins dispersed in a medium with a pH gradient
along the direction of an applied electric field
• Each protein in the mixture will stop moving at a position in the gradient where the
pH is equal to the isoelectric point
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Support Media
Paper Electrophoresis
• One of the first matrices used for electrophoresis
• In paper electrophoresis, the sample is applied directly to a zone on
the dry paper, which is then moistened with a buffer solution before
application of an electric field
• Dyes are combined with samples and standards to help visualize the
progress of the electrophoresis
• The movement of samples on paper is best when the current flow is
parallel to the fiber axis in the paper
• Some advantages of paper are that it is readily available and easy to
handle, requires no preparation, and allows the rapid development of
new methodologies
• Besides being easy to obtain, paper does not contain many of the
bound charges that can interfere with the separation
• A disadvantage of paper electrophoresis is that the porosity of
commercial paper is not controlled, and therefore the technique is
not very sensitive, nor is it easily reproducible
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Support Media
Polyacrylamide Gels
• One of the most commonly used electrophoretic methods
• Analytical uses of this technique center on protein nucleic
acid characterization (e.g. purity, size, or molecular weight,
and composition)
• Acrylamide is neurotoxin, however, the reagents must be
combined extremely carefully
• The sieving properties of the gel are defined by the
network of pores established during the polymerization : as
the acrylamide concentration of the gel increases, the
effective pore size decreases
• The most commonly used combination of chemicals to
produce a polyacrylamide gel is acrylamide, bisacrylamide,
buffer, ammonium persulfate, and tetramethylenediamine
(TEMED)
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Agarose Gel
Support Media
• Agarose is a polymer extracted from red seaweed
• When agar is extracted from the seaweed, it is in two components, agaropectin
and agarose
• The agarose portion is nearly uncharged, making it desirable for use as on
electrophoresis matrix
• The advantages of agarose electrophoresis are that it requires no additives or
cross-linkers for polymerization, it is not hazardous, low concentration gels are
relatively sturdy, and it is inexpensive
• Commonly used for the separation of large molecules such as DNA fragments
Capillaries
• The fused silica capillaries are flexible due to an outer polyimide coating and are
available in inner diameters ranging from 10 to 300 µm
• Fused silica is transparent to UV light, which enables the capillary to serve as its
own detection flow cell
• Electrostatic interactions with the capillary surface can develop, however, when
charged species are being separated
• To overcome this problem is to chemically modify the inner capillary surface to
produce a nonionic, hydrophilic coating, resulting in the shielding of the silanol
functionalities
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Support Media
Comparison of Electrophoresis Matrices
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Detection Techniques
Chemical Staining
• Incorporate a “fixing” step, such as a soak in dilute acetic acid for 1 h,
etiher before or in conjunction with staining.
• Frequently used stains: Coomassie brilliant blue (R250 and G250) and
silver stain
• The gel then is scanned with densitometer
Fluorescence
• Provides much better detection limit than simple chemical stains,
typically involves the covalent binding of the fluorescent residue to
the analyte
• Fluorescamine- popular reagent for labeling of proteins
• At room temp. and alkaline pH, fluorescamine can react with primary
amine on the protein to generate a fluorescent derivative
• The reagent ethidium bromide often used to visualize DNA
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Detection Techniques
Radioactivity
• If a sample is radioactive, the bands that separate during
electrophoresis are subsequently radioactive
• When the separation is complete, the electrophoretic matrix can be
placed against x-ray film until the radiation makes a mirror image of
the banding pattern on the film
Immunoelectrophoretic Techniques
• Known as “crossed immunoelectrophoresis”
• A sample is first run longitudinally through an agarose gel for a
predetermined time
• Second, a longitudinal strip of the gel area in which of the sample was
electrophoresed is typically cut out and placed into a similarly sized
area of an antibody containing gel
• As an electric current is applied to the gel, each band of the sample
with form an antigen-antibody precipitation pattern through the gel
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