Lecture 4
Bio 451 Molecular Biology Techniques
Electrophoresis
History of electrophoresis
1930s First reports of the use of
sucrose for gel electrophoresis.
1955 Introduction of starch gels,
not very good separation
1959 Introduction of acrylamide
gels for 1st time (Raymond and Winstraub) Accuracy of control of
parameters such as pore size and stability
1964 Disc gel electrophoresis
Ornstein and Davis
1969 Introduction of denaturing
agents especially SDS separation of protein subunit (Beber and Osborn)
1970 Laemmli-T4 phage separated 28
components “stacking gel + SDS
1975 2 Dimensional gels
O’Farrell isoelectric focussing then SDS gel electrophoresis.
1977 Sequencing gels
Late 1970s agarose gels
1983 Pulsed field gel
electrophoresis
1983 Capillary electrophoresis
introduced
Electrophoretic Process
·
Two
pieces of equipment are necessary for electrophoresis: a d.c. power supply and
a buffer reservoir system.
·
The
system consists of upper and lower buffer reservoirs with provision for
suspending the gel. The only
electrical connection between the reservoirs is via the gel. Platinum electrodes are positioned in
each reservoir.
·
The
macromolecular samples are dissolved in a glycerol containing buffer to
increase the density of the sample.
At pH 9, a commonly used pH for electrophoresis, most proteins are
negatively charged. Hence the
anode is placed in the lower reservoir.
Often, a “tracking dye” such as bromophenol blue (BB) is
included in the sample as a reference.
BB migrates faster than any of the macromolecules.
·
The
reaction that permit current passage from the cathode to the anode are the
electrolysis of water, producting hydrogen at the cathode and oxygen at the
anode:
Cathode reaction: 2e-
+ 2H2O – 2 OH- + H2
HA
+ OH- --- A- + H2O
Anode reaction: H2O
--- 2H+ + 1/2 O2 + 2e-
H+
+ A- --- HA
One mole of hydroxyl ions and protons are produced at the
cathode and anode, respectively, for every mole of electrons that flow through
the system.
Types of Electrophoresis
Low voltage
thin sheet electrophoresis
· The supporting
medium may be paper, cellulose acetate, or a thin layer of material such as
silica.
· Paper. Chromatography paper is suitable for
electrophoresis. Low voltage paper
electrophoresis has been extensively used in the past for the separation of a
range of charged compounds such as amino acids, peptides, proteins,
nucleotides, nucleic acids and charged carbohydrate derivatives. Considerable diffusion of small
molecules occurs on paper during low voltage electrophoresis and better
resolution may be obtained by applying high voltage, where the time required
for separation is reduced and less diffusion of the molecules occurs.
· Cellulose Acetate. Cellulose acetate is a suitable medium
for the separation of radiolabelled substances and for such microtechniques as
immunodiffusion and immunoeletrophoresis.
Cellulose acetate will, in general, separate the same range of compounds
as paper but has found particular application in clinical investigations for
the separation of blood proteins, including glycoproteins, lipoproteins and
hemoglobins.
· Thin Layer Electrophoresis (TLE). Thin layers of silica, alumina or
cellulose can be prepared on glass plates as for thin layer chromatography
(TLC). The plates are placed horizontally into the electrophoresis unit and the
thin layer is allowed to saturate with buffer by diffusion from the
reservoir. TLE, like TLC, is rapid
and gives good resolution and high sensitivity. High voltages may also be used in TLE.
Gel
Electrophoresis
· Gels may be
prepared from starch, agar and polyacrylamide. The molecular sieving property of the semi-rigid gel helps
to separate large ionic compounds such as proteins. Smaller molecules can be separated only in Sephadex-type
gels. Sephadex-type gels have
small pores that exclude larger molecules from access to the stationary phase
inside the particle, thereby causing movement outside the pores whereas small
molecules are tightly held within the pores.
· Starch gels are prepared by
heating and cooling a mixture of partially hydrolysed starch in an appropriate
buffer. This causes the branched
chains of the amylopectin components of starch to intertwine and form a
semi-rigid gel.
· Agar/Agarose gels consist of two
galactose-based polymers, agarose and agaropectin.
Electrophoresis: Acrylamide Gel Electrophoresis
· Polyacrylamide gels are made
from acrylamide monomers copolymerised with the cross linker
N,N’methylenebisacrylamide in the presence of ammonium persulphate and
TEMED as catalyst. Prior degassing
of solutions is required since molecular oxygen inhibits chemical
polymerization.
The Tools of Biochemistry by Terrance G. Cooper, Univ. of Pittsbrugh, Wiley-Interscience Publications, John Wiley and Sons, NY, 1977.
Make up of the Acrylamide gel
The
migration of macromolecules through acrylamide gel is influenced by the
structure of the gel itself.
·
The
compounds used to construct the polymer matrix are acrylamide,
N,N’-methylene-bis(acrylamide), tetramethylenediamine (TEMED), and
ammonium persulfate (ammonium persulfate dissolved in water it forms free
radicals).
·
If the
ammonium persulfate free radicals are brought into contact with acrylamide, the
free radical is retained within the acrylamide molecule. The “activated” acrylamide
can then react with successive acrylamide molecules producing a polymer chain.
To obtain a gel, the acrylamide needs to be cross-linked with
N,N’-methylene-bis(acrylamide).
TEMED or b-(dimethylamino) propionitrile is usually added at at
concentration of 0.4% to serve as a catalyst of gel formation because of its ability
to exist in a free radical form.
·
The
size of the holes or pores in the gel is determined by two parameters: (1) the
amount of acrylamide used and (2) the degree of cross-linkage. The average pore size reaches a minimum
when 5% of the total acrylamide used is
N,N’-methylene-bis(acrylamide).
Therefore, in many formulations the bis(acrylamide) content is fixed at
5% of the total acrylamide and is not altered. In the lab, we used 30% Acryl+0.8% Bis (2.6% bis). The pore
size is manipulated by varying the total content of acrylamide.
The
following are other considerations in the production of polyacrylamide
gels.
·
The
buffer serves (1) to maintain a constant pH within the reservoirs and within
the acrylamide gel and (2) as the electrolyte which conducts current across the
electric field.
·
A
buffer must be chosen that does not interact with the macromolecules being
separated. Such interactions might
change the rate of molecule migration. This would occur if the interaction adds
or neutralizes the charge on the macromolecule.
·
The pH
of the environment must allow the macromolecules to be charged not
denatured. In the case of proteins
the usual outside limits are 4.5 and 9.0.
However, a broader or more restricted range may be necessary.
·
The
ionic strength and concentration of the buffer must be considered. If the electrolyte concentration within
the acrylamide gel is too low, the migrating macromolecules conduct a large
portion of the current. As a
result they are not found in a sharp band but rather spread into diffuse zones
and thus greatly decrease the resolution of this method. Alternatively, if the electrolyte
concentration within the gel is too high, the amount of current conducted
increases while voltage decreases.
This usually results in a decreased rate of macromolecular migration and
the generation of heat, which can denature the proteins being separated.
· The extent to which a macromolecule
migrates into the gel depends on both the size of the molecules and the average
size of the pores thorough which it must pass. It is this sieving effect that enhances the resolution
obtained with acrylamide gels.
Logarithms of the relative mobilities for a group of proteins decrease
linearly as the total gel concentration is increased.
Discontinuous Gel Electrophoresis
·
Disc
pH or disc electophoresis is a modification of the zone electrophoretic
technique described above. The
significant differences are (1) the use of a two gel system and (2) the unique
buffer systems used in the gel matrix and in the buffer reservoirs. The lower-separating or running gel is
prepared using about the same amount of acrylamide (5-10%) as would be used for
an analogous zone electrophoretic experiment. The buffer used in this gel is usually an amine such as Tris
which is adjusted to the proper pH (e.g., pH 8.8) using hydrochloric acid.
·
After
the separating gel has polymerized, a second, small layer of gel is polymerized
on top of it. This is the upper or
stacking gel. A total acrylamide
content of 2-3% acrylamide is common.
The buffer used in the stacking gel is also an amine such as Tris, but
in this case the pH is adjusted with HCl to 2 pH units lower than that of the
running gel (pH 6.8).
·
The
buffer used in the protein sample should be identical to that used in the stacking
gel.
·
The
reservoir buffer is made up of 0.025M Tris, 0.192 M Glycine, pH 8.8).
·
Glycine
exists as both a zwitterion with a net charge of zero and a glycinate ion with
a charge of minus one:
NH3CH2COO- ------
NH2CH2COO- + H+
·
When
the electric field is established, chloride, protein, bromophenol blue, and
glycinate anions all begin to migrate toward the anode. However, as glycinate ions enter the
sample buffer and stacking gel they encounter a low pH, shifting the
equilibrium toward formation of zwitterions, which are immobile. Failure of glycine zwitterions to move
into the sample and stacking gel creates a deficiency of mobile ions, which in
turn decreases current flow.
However, a constant current must be maintained throughout the entire
electrical system. This is
accomplished in the area between the leading chloride ions and the trailing
glycinate ions by an increase in voltage.
The result is a very high localized voltage gradient occurring between
the chloride ions and the glycinate ions.
In this condition the relative ion mobilities are glycinate<
proteins<BB<Cl-.
·
In
this strong local electric field the anionic proteins all migrate rapidly. The stacking gel has large pores so as
not to impede their progress. If
any of the proteins overtake the leading chloride ions, they slow down because
wherever there are chloride ions there is no ion deficiency, and hence the
large field strength disappears.
The rapid movement of the proteins behind the chloride front and the
decreased rate of protein migration as they approach the front result in a
piling up or concentration of the protein sample in a tight disc between the
glycinate and chloride ions. As
the disc of proteins encounters the running gel their migration is slowed by
the small pores of the gel. This
permits the small glycinate ions to catch up with the proteins. Crossing the interface between the
stacking and running gels, glycinate ions become fully charged once again, and
there is no longer an ion deficiency.
Hence from this point on there is a constant field strength throughout
the gel, and separation of the protein proceeds just a with zone
electrophoresis. The advantage of
this modification is that the protein sample enters the separating gels as a
narrow zone. The resulting protein
bands are therefore much more compact, and this increases resolution.
SDS Acrylamide Gel Electrophoresis
·
Molecules
which are tightly, but not covalently, bound together do not usually separate
from one another during electrophoresis.
Thus, the anionic detergent sodium dodecyl sulfate (SDS) is used to
separate a mixture of proteins.
SDS binds to the hydrophobic regions of proteins and separates most of
them into their component subunits.
1.4g SDS binds to 1 gram of protein. SDS binding also imparts a large negative charge to the
denatured, randomly coiled polypeptides.
This charge largely masks any charge normally present in the absence of
SDS.
·
If
component subunits of protein are held together by disulfide bonds, these bonds
may be broken before electrophoresis by heating the preparation in the presence
of SDS and b-mercaptoethanol, a reducing agent.
Two
Dimensional Gel Electrophoresis
·
This
powerful technique involves first subjecting the mixture of proteins to
isoelectric focusing on a 1 mm diameter gel in a capillary tube. Isoelectric focusing is carried out in much
the same way as elecrtrophoresis with the exception that ampholytes are
electrophoresed through the gel prior to sample addition in order to establish
a pH gradient within it. As a
protein moves down the tube it continuously encounters a higher and higher pH
environment. As the protein
traverses this pH gradient it reaches a pH corresponding to its isoelectric
point. At this pH the net charge
of the protein is zero and it therefore stops moving in the electric
field.
·
At the
end of isoelectric focusing, the gel is removed from the capillary tube and
placed on top of a slab gel. The
sample is now subjected to SDS acrylamide gel electrophoresis. Since the isoelectric point and
molecular weight of a protein are unrelated it is possible to obtain an even
distribution of proteins using these parameters in two dimensions.
·
Gradient
Gel Electrophoresis
· Isoelectric focusing. This technique, also called
electrofocusing is based on moving boundary rather than zone
electrophoresis. Amphoteric
substances such as amino acids and peptides are separated in an electric field
across which there are both voltage and pH gradients. The anode region is at a lower pH than the cathode region
and a stable pH gradient is maintained between the electrodes. Amphoteric substances thus become
focused into narrow stationary bands.
The carrier ampholytes are usually synthetic aliphatic
polyamino-polycarboxylic acids and are available commercially in mixtures
covering a wide pH band (e.g. 3 to 10) or various narrow bands (e.g. 4 to
5). The anode end of the column is
connected to a reservoir containing an acid solution (e.g. phosphoric acid) and
the cathode end is connected to a reservoir containing an alkaline solution
(e.g. sodium hydroxide). On
opening the two reservoir valves the two solutions diffuse into the column
setting up a pH gradient between the acidic anode and the alkaline
cathode. The valves are then
closed and the current switched on, causing the carrier ampholytes to migrate
until they reach the pH regions where they have no net charge. The sample is then allowed to
enter the upper end of the column by opening a valve, and the charged sample
component migrate until they reach the pH regions of the tube at which they
have no net charge. Vertical columns containing polyacrylamide gel impregnated
with carrier ampholytes can also be used, reducing the time required for
separation.
· Isotachophoresis. This technique is another form of moving
boundary electrophoresis. The
principle underlying isotachophoresis is utilized in the stacking gel system in
SDS polyacrylamide electrrophoresis.
In greek, isotachophoresis means travel (phoresis) at the same (iso)
speed (tacho). Any charged
substances can be separated by isotachophoresis. For the separation of a mixture of anions, a leading anion
(e.g. Cl-) is chosen which has a higher mobility than the sample
ions, and a trailing anion (e.g. glutamate) is also chosen which has lower
mobility than the sample ions. All
the anions must have a common cation (e.g. Tris). When the current is switched on, the leading ions will move
towards the appropriate electrode, the sample ions will follow in order of
their mobilities and the trailing ion will follow behind the sample ions.
· Pulse-Field Gel Electrophoresis. When a DNA molecule is larger than the
pore size, the DNA can no longer be sieved by the gel according to its size but
must instead migrate “end-on” through the matrix as if through a
sinuous tube. This mode of migration
is known as “reptation.” Gels of 0.1-0.2% agarose can resolve
extremely large DNA molecules, but they are very fragile. Even then, these gels are incapable of
resolving linear DNA molecules larger than 750kb. DNA molecules in the individual chromosomes of lower
eukaryotes may be 7000 kb or more in length.
· In 1984, Schwartz
and Cantor reported the development of pulse-field gel electrophoresis. In this method, pulse, alternating,
orthogonal electric fields are applied to a gel. Large DNA molecules become trapped in their reptation tubes
every time the direction of the electric field is altered and can make no
further progress throughout the gels until they have reoriented themselves
along the new axis of the electric field.
The larger the DNA molecules, the longer the time required for this
realignment. Molecules of DNA
whose reorientation times are less than the period of the electric pulse will
therefore be fractionated according to size. The original method described by Schwartz and Cantor was
capable of resolving DNAs up to 2 megabase pairs in length. However, as a consequence of
improvements to the technique, resolution of DNA molecules larger than 5000kb
can now be achieved.
· In 1986, Gardiner
et al used a vertical gel apparatus with platinum wire electrodes positioned on
opposite sides of the gel. The DNA
moves first toward one set of electrodes and then toward the other as the
electric fields are switched. The
net result of these zigzag movements is a straight line from the loading well
toward the bottom of the gel.
Because all the lanes in the gel are exposed to equivalent electric
fields, there is no horizontal distortion of the DNA bands. As with other pulsed-field gel
electrophoresis systems, the size of the molecules resolved at a given voltage
is a function of the angle between the electrodes and the pulse time. Typically, the electrodes are arranged
at a 90o angle and 10 second pulses are used to separate molecules
between 50 kb and 450kb in length; 50 to 60-second pulses are used to
fractionate molecules larger than 1000 kb in length. The upper limit of resolution of the system appears to be
approx. 9000kb.
Detection
of Macromolecules
·
Coomassie
Brilliant Blue Staining for proteins
·
Alcian
blue, Toluidine blue for glycoproteins
·
Amido
black, Wilson sudan black for lipoproteins
·
Fluorescent
Staining Techniques: Pluorescamine
·
Specific
Enzyme Visualization: lactate dehydrogenase-reduction of nitroblue tetrazolium
chloride (yellow to blue); Periodic acid-Schiff for glycoproteins.
· Detection of Radioactive
Macromolecules: X-rays, liquid scintillation, sepctrophotometry.
Capillary Electrophoresis (CE). This type of electrophoresis can separate
a wide variety of molecules of biological interest such as metabolites, drugs,
amino acids, nucleic acids, carbohydrates, peptides and proteins based on their
sizes and ionic properties. CE
separations of proteins and peptides are based on charge-to-mass ratios. While PAGE separations are restricted
to polyacrylamide matrices and a relatively small number of buffer systems, CE
separations can be achieved in a variety of matrices and buffers. Consequently, there is much greater
flexibility in the design of the optimal separation protocols.
· CE employs as
fused-silica capillary column, which may or may not be derivatized, either in
free solution or in the presence of a fluid matrix. The CE separation is more analogous to HPLC than PAGE. However, CE separation is based on
electrophoretic parameters. CE is
most useful for separations of peptides because it offers great flexibility in
separation parameters. It can be
used to monitor proteolytic digestions and optimize digestion conditions for
the production of a representative peptide fingerprint of a protein.
· The capillaries
are generally 30 to 50 cm long with 50-75 µm internal diameter. The net total volume of these
capillaries is in the low microliter range. The capillaries are thin-walled; this allows rapid and
efficient exchange of the Joule heating that results from the high voltages
that are necessary for electrophoretic separations. The maximum Voltage and current that can be run are
300µA and 30kV. As the
protein and peptide molecules are swept through the capillary, they pass
through the detector light path and are registered on the UV monitor. With respect to sensitivity, speed, and
versatility, CE can offer some advantages over gel electrophoresis for the
separation of proteins and peptides.
· In CE, buffer
flow is generated inside the column when the electric field is applied. This flow is from the cathode electrode
to the anode electrode in a a fused-silica capillary column; this movement of
the buffer is called the electroosmotic flow (EOF)
· Isoelectric
focusing can be performed by CE. A
pH gradient is generated in a coated capillary column by filling the column
with asample solution that contains ampholytes. A high pH solution (NaOH) is placed in the cathode reservoir
and a low pH solution (phosphoric acid) is placed in the anode reservoir. After separation by IEF in the absence
of electroosmotic flow, mobilization of the proteins past the detector is
necessary. A simple replacement of
the anode reservoir with the buffer in the cathode reservoir established EOF
and causes the sample zone to migrate pass the detector.
· The
selection of an appropriate separation protocol for a protein depends on the
specific properties of that protein.
However, there are separation approaches that utilize specific
properties of proteins. IEF-CE
separates on the basis of charge alone; CE in the presence of ionic detergents
such as SDS separates on the basis of size alone; and CE using underivatized
capillaries at low pH or derivatized capillaries at high salt and high pH
separates on the basis of both size and charge.
Factors affecting electrophoresis
The electric
field
· Voltage. If the separation of the electrodes is d (cm) and the potential difference
between them is V volts, the
potential gradient is V/d. The
force on an ion bearing a charge q coulombs is then Vq/d newtons. The
force causes migration and the rate of migration is proportional to Vq/d. The rate of migration under unit potential gradient is
called the mobility of the ion. An
increase in the potential gradient will therefore increase the rate of
migration proportionally.
· Current. When a potential
difference is applied between the electrodes, a current is generated, measured
in coulombs sec or amperes. The
size of this current is determined by the resistance of the medium and is
proportional to the voltage. The
current in the solution between the electrodes is conducted mainly by the buffer
ions with a small proportion being conducted by the sample ions. An increase in voltage will increase
the total charge per second conveyed towards the electrode. The distance migrated by the ions will
be proportional to both current and time.
· Resistance. Ohm’s law expresses the
relationship between current I (measured in amperes, A), voltage V (measured in volts, V) and resistance R (measured in ohms, W) in which:
V/I=R
· The current, and
hence the rate of migration are thus inversely proportional to the resistance,
which in turn is a function of the medium, the buffer and its
concentration. Resistance will
increase with the length of the supporting medium but will decrease with its
cross-sectional area and with increasing buffer ion concentration. During electrophoresis the power
dissipated in the supporting medium (W, measured in watts) is such that:
W=I2R
· An increase in
temperature will cause the resistance to fall. Part of this effect is due to an increase in the mobility of
the ions as a result of a decrease in the viscous resistance offered by the
liquid to the motion of the ions through it as the temperature rises. The heating will produce evaporation of
the solvent from the supporting medium causing a decrease in resistance. Although the rate of migration and the
total charge per second conveyed towards the electrode will increase, the
increase in buffer ion concentration will result in slower migration of the
sample.
· When constant
voltage is applied, the current will increase during electrophoresis due to
a decrease in resistance of the medium with the rise in temperature. Consequently, more heat will be
produced resulting in more evaporation of solvent and a decrease in
resistance. A constant current
avoids these problems but may lead to a drop in voltage due to decreased
resistance, resulting in reduced rate of migration.
· If a number of
gels are run in parallel with one power supply, then:
1/R= 1/r1
+1/r2+…. 1/rn
where R is the
total resistance and r1,r2, etc. are the resistances of
each gel. The total current
supplied must be increased in proportion to the number of gels used, assuming
that they all have the same resistance.
The resistance does decrease
as the temperature increase for semiconductors. Since the gel is a semiconductor so it make sense
for the decrease in resistance.
This is because increasing temperature means increasing the
velocity of charged particles so conductivity will
increase. However, the resistance in metals does increase as
the temperature increase. When the temperature of a metal is
increased its resistance also increases this is because the increased vibration
of the matel lattice makes it harder for the free electrons to flow easily
through the material. When a
semiconductor is heated its resistance goes down as more electrons have the
energy to become free from the lattice.
The sample
· Charge. The rate of migration increases with an
increase in the net charge. The
magnitude of the charge is generally pH dependent.
· Size. The rate of migration decreases for larger molecules, due to
the increased frictional and electrostatic forces exerted by the surrounding
medium.
· Shape. Molecules of similar size but different
shapes such as fibrous and globular proteins exhibit different migration
characteristics because of the differential effect of frictional and
electrostatic forces.
The buffer
· Composition. The buffers in common use are formate,
acetate, citrate, barbitone, phosphate, Tris, EDTA and pyridine. The buffer should not bind to the
sample. In some cases, however,
binding can be advantageous, for example borate buffers are used to separate
carbohydrates since they produce charged complexes with carbohydrates. Since the buffer acts as a solvent for
the sample, some diffusion of the sample is inevitable, being particularly
noticeable for small molecules such as amino acids and sugars. The extent of diffusion can be
minimized by not overloading the sample.
· Concentrations As the ionic strength of the buffer
increase, the proportion of current carried by the buffer will increase and the
share of the current carried by the sample will decrease, thus slowing the
sample migration. High ionic
strength of the buffer will also increase the overall current and heat will be
produced. At low ionic strengths
the proportion of current carried by the buffer will decrease and the share of
the current carried by the sample will increase, thus increasing its rate of
migration. A low ionic buffer
strength reduces the overall
current and results in less heat production, but diffusion and the
resulting loss of resolution are higher.
The compromise is to use between 0.05 to 0.1 M.
· PH determines extent of ionisation.
The supporting
medium
· Adsorption. This is the retention of sample
molecules by the supporting medium.
Adsorption causes tailing of the sample and reduces the rate and
resolution of the separation.
· Electro-osmosis. This results from a relative charge
being produced between water molecules in the buffer and the surface of the
supporting medium. The charge may
be caused by surface adsorption of ions from the buffer and the presence of
stationary carboxyl groups on paper or sulphonate groups on agar. The effects of electro-osmosis can
normally be ignored.
· Molecular Sieving. Molecular sieving occurring in agar,
starch and polyacrylamide gels is that the movement of large molecules is
hindered increasingly by decreasing the pore size since all molecules have to
traverse through the pores.