GEL ELECTROPHORESIS

SINGH NISHANT
Roll no: 29
Under the guidance of R.M. Mashru
INTRODUCTION
• Positive or negative electrical charges are frequently associated with
biomolecules. When placed in an electric field, charged biomolecules
move towards the electrode of opposite charge due to the phenomenon
of electrostatic attraction.
• Electrophoresis is the separation of charged molecules in an applied
electric field. The relative mobility of individual molecules depends on
several factors. The most important of which are net charge, charge/mass
ratio, molecular shape and the temperature, porosity and viscosity of the
matrix through which the molecule migrates.
• Complex mixtures can be separated to very high resolution by this
process
Gel types
• AGAROSE:
• The most widely used polysaccharide gel matrix nowadays is that formed with
agarose. This is a polymer composed of a repeating disaccharide unit called
agarobiose which consists of galactose and 3,6-anhydrogalactose (Fig. 1).
• Agarose gives a more uniform degree of porosity than starch and this may be
varied by altering the starting concentration of the suspension (low
concentrations give large pores while high concentrations give smaller pores).
• This gel has found wide spread use especially in the separation of DNA
molecules (although it may also be used in some electrophoretic procedures
involving protein samples such as immuno-electrophoresis). Because of the
uniform charge distribution in nucleic acids, it is possible accurately to
determine DNA molecular masses based on mobility in agarose gels. However
the limited mechanical stability of agarose, while sufficient to form a stable
horizontal gel, compromises the possibilities for post-electrophoretic
manipulation.
• ACRYLAMIDE:
• A far stronger gel suitable for electrophoretic separation of both
proteins and nucleic acids may be formed by the polymerization of
acrylamide. The inclusion of a small amount of bisacrylamide leads
cross linking by a methylene bridge (N,N′ methylene diacrylamide)
• Therefore forming a cross linked gel with a highly-controlled porosity
which is also mechanically strong and chemically inert.
• For separation of proteins, the ratio of acrylamide : N,N′ methylene
diacrylamide is usually 40:1 while for DNA separation it is 19:1.
• Such gels are suitable for high-resolution separation of DNA and
proteins across a large mass range.
• Bisacrylamide means two molecules of acrylamide are connected
through a methylene bridge.
•

• free radicals

• NH4SO4 -acrylamide
• Decomposition

• TEMED -bisacrylamide
Generation of free radicals are exothermic rexn , as this results in
warming up the gel and liberates bubbles . Degassing is necessary prior.
• Another method is photopolymerization
• In this TEMED and ammonium sulphate are replaced by riboflavin
which generates free radicals by photo degradation
Staining of gel
• One of the most important aspects of gel electrophoresis technique is
staining.
• Once sample molecules have separated in the gel matrix it is
necessary to visualize their position.
• This is achieved by staining with an agent appropriate for the sample.
Some of the more common staining methods used in biochemistry
are listed in Table 1.
Preparation and running of Std electrophoresis

• The equipment and supplies necessary for conducting agarose gel

electrophoresis are relatively simple and include:
• An electrophoresis chamber and power supply
• Gel casting trays, which are available in a variety of sizes and composed
of UV-transparent plastic. The open ends of the trays are closed with
tape while the gel is being cast, then removed prior to electrophoresis.
• Sample combs, around which molten medium is poured to form sample
wells in the gel.
• Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-
EDTA (TBE).
• Loading buffer, which contains something dense (e.g. glycerol) to
allow the sample to “fall” into the sample wells, and one or two
tracking dyes, which migrate in the gel and allow visual monitoring or
how far the electrophoresis has proceeded.
• Staining: DNA molecules are easily visualized under an ultraviolet
lamp when Electrophoresed in the presence of the extrinsic fluor
ethidium bromide.
• Alternatively, nucleic acids can be stained after electrophoretic
separation by soaking the gel in a solution of ethidium bromide.
• When intercalated into double-stranded DNA, fluorescence of this
molecule increases greatly. It is also possible to detect DNA with the
extrinsic fluor 1-anilino 8-naphthalene sulphonate.
• Transilluminator (an ultraviolet light box), which is used to visualize
stained DNA in gels. NOTE: always wear protective eyewear when
observing DNA on a Transilluminator to prevent damage to the eyes from
UV light. Fig. 2. Preparation, loading and running of gel in electrophoresis.
• To prepare gel, agarose powder is mixed with electrophoresis buffer to
the desired concentration, and heated in a microwave oven to melt it.
• Ethidium bromide is added to the gel (final concentration 0.5 ug/ml) to
facilitate visualization of DNA after electrophoresis.
• After cooling the solution to about 60°C, it is poured into a casting tray
containing a sample comb and allowed to solidify at room temperature.
• After the gel has solidified, the comb is removed, taking care not to rip
the bottom of the wells. The gel, still in plastic tray, is inserted horizontally
into the electrophoresis chamber and is covered with buffer.
• Samples containing DNA mixed with loading buffer are then pipetted
into the sample wells, the lid and power leads are placed on the
apparatus (Fig. 2), and a current is applied. The current flow can be
confirmed by observing bubbles coming off the electrodes. DNA will
migrate towards the positive electrode, which is usually colored red,
in view of its negative charge.
• The distance DNA has migrated in the gel can be judged by visually
monitoring migration of the tracking dyes like bromophenol blue and
xylene cyanole dyes.
Agarose gel electrophoresis of DNA
• MIGRATION OF DNA FRAGMENTS IN AGAROSE
• Fragments of linear DNA migrate through agarose gels with a mobility
that is inversely proportional to the log10 of their molecular weight.
• In other words, if you plot the distance from the well that DNA
fragments have migrated against the log10 of either their molecular
weights or number of base pairs, a roughly straight line will appear.
• AGAROSE GEL POLYACRYLAMIDE

• Agarose gels contains larger pores than acrylamide gels comparatively.

• Still very large molecules can get stuck in agarose gel also hence selection
criteria becomes an imp factor here [ gel must contain appropriate size
pores to allow DNA fragment to pass…
• Circular forms of DNA migrate in agarose distinctly differently from
linear DNAs of the same mass.
• Typically, uncut plasmids will appear to migrate more rapidly than the
same plasmid when linearized.
• Additionally, most preparations of uncut plasmid contain at least two
topologically-different forms of DNA, corresponding to supercoiled
forms and nicked circles .
• Several additional factors have important effects on the mobility of
DNA fragments in agarose gels, and can be used to your advantage in
optimizing separation of DNA fragments. Chief among these factors
are:
• DNA size is represented in kilobase pairs(kbp).
• As earlier said , all fragments have equal negative charge , so the
separation is on the basis of molecular weight , as resistance offered
by the agarose gel.

• The porosity of gel depend on the concentration of agarose used.

• Higher concentration means lower will be the pore size and vice versa.
• Since agarose gel separate DNA on basis of size , the Mr of the DNA
fragments may be easily determined by running the no of std DNA
markers of known Mr on same gel.
• This is generally achieved by running cleaved bacteriophage DNA of
known Mr as a std .
 0.3% 0.8% 2.0%
•0.5-
•5-60kb •0.1-3kb
10kb
• AGAROSE CONCENTRATION: By using gels with different
concentrations of agarose, one can resolve different sizes of DNA
fragments. Higher concentrations of agarose facilitate separation of
small DNAs, while low agarose concentrations allow resolution of
larger DNAs.
• VOLTAGE: As the voltage applied to a gel is increased, larger
fragments migrate proportionally faster those small fragments. For
that reason, the best resolution of fragments larger then about 2 kb is
attained by applying no more than 5 volts per cm to the gel (the cm
value is the distance between the two electrodes, not the length of
the gel).
Add agarose into loading buffer and heat it to dissolve it . Surround it by
adhesive tape and plastic frames depth must be 3mm.
Loading wells are casted with the help of combs , remove it later.
Prepare the sample by dissolving it into loading buffer with glycerol and sucrose
to give it viscosity so it will sink in tank. Gel is prior place in tank and add
electrophoresis buffer . Inject the samples into loading wells. No additional
stacking gel layer required because mobility of dna is much greater in well as
compared to gel.
A dye such as bromophenol blue is also included in the sample solvent; it makes
it easier to see the sample that is being loaded and also acts as a marker of the
electrophoresis front.
General purpose gels are approximately 25 cm long and 12 cm wide and are run
at a voltage gradient of about 1.5 V cm-1 overnight.
A higher voltage would cause excessive heating. For rapid analyses that do not
need extensive separation of DNA molecules, it is common to use mini-gels that
are less than 10 cm long . In this way information can be obtained in 2-3 h.
• ELECTROPHORESIS BUFFER: Several different buffers have been
recommended for electrophoresis of DNA.
• The most commonly used for duplex DNA are TAE (Tris-acetate-EDTA)
and TBE (Tris-borate-EDTA). DNA fragments will migrate at somewhat
different rates in these two buffers due to differences in ionic
strength.
• Buffers not only establish a pH, but provide ions to support
conductivity. If you mistakenly use water instead of buffer, there will
be essentially no migration of DNA in the gel!
• Conversely, if you use concentrated buffer (e.g. a 10X stock solution),
enough heat may be generated in the gel to melt it.
• EFFECTS OF ETHIDIUM BROMIDE[MARKER]: Ethidium bromide is a
fluorescent dye that intercalates between bases of nucleic acids and
allows very convenient detection of DNA fragments in gels, under
ultraviolet light DNA observed as orange red colored.
• As described above, it can be incorporated into agarose gels, or added
to samples of DNA before loading to enable visualization of the
fragments within the gel.
• As might be expected, binding of ethidium bromide to DNA alters its
mass and rigidity, and therefore its mobility.
ELECTROPHORESIS OF PROTIEN
• Sodium dodecyl sulphate (SDS)–polyacrylamide gel
electrophoresis
• SDS–polyacrylamide gel electrophoresis (SDS–PAGE) is the most widely
used method for analyzing protein mixtures qualitatively.
• It is particularly useful for monitoring protein purification and, because the
method is based on the separation of proteins according to size, it can also
be used to determine the relative molecular mass of proteins.
• Samples to be run on SDS–PAGE are firstly boiled for 5 min in sample
buffer containing b-mercaptothion and SDS.
• The mercaptothion reduces any desulphated bridges present that are
holding together the protein tertiary structure, and the SDS binds strongly
to, and denatures, the protein.
Fig: A typical SDS–polyacrylamide gel. All 10 wells in the gel have
been loaded with the same complex mixture of proteins
• Each protein in the mixture is therefore fully denatured by this
treatment and opens up into a rod-shaped structure with a series of
negatively charged SDS molecules along the polypeptide chain.
• On average, one SDS molecule binds for every two amino acid
residues. The original native charge on the molecule is therefore
completely swamped by the negatively charged SDS molecules.
• The rod-like structure remains, as any rotation that tends to fold up
the protein chain would result in repulsion between negative charges
on different parts of the protein chain, returning the conformation
back to the rod shape.
• The sample buffer also contains an ionizable tracking dye, usually
bromophenol blue, that allows the electro-phoretic run to be
monitored, and sucrose or glycerol, which gives the sample solution
density thus allowing the sample to settle easily through the
electrophoresis buffer to the bottom when injected into the loading
well.
• Once the samples are all loaded, a current is passed through the gel.
The samples to be separated are not in fact loaded directly into the
main separating gel.
• When the main separating gel (normally about 5 cm long) has been
poured between the glass plates and allowed to set, a shorter
(approximately 0.8 cm) stacking gel is poured on top of the separating
gel and it is into this gel thet the wells are formed and the proteins
loaded.
• The purpose of this stacking gel is to concentrate the protein sample into a
sharp band before it enters the main separating gel. This is achieved by utilizing
differences in ionic strength and pH between the electrophoresis buffer and the
stacking gel buffer and involves a phenomenon known as utilizing.
• The stacking gel has a very large pore size (4% acrylamide), which allows the
proteins to move freely and concentrate , or stack, under the effect of the
electric field. The band-sharpening effect relies on the fact that negatively
charged glycinate ions (in the electrophoresis buffer) have a lower
electrophoretic mobility then do the protein–SDS complexes, which, in turn,
have lower mobility than the chloride ions (Cl- of the loading buffer and the
stacking gel buffer
• When the current is switched on, all the ionic species have to migrate at the
same speed otherwise there would be a break in the electrical circuit. The
glycinate ions can move at the same speed as Cl- only if they are in a region of
higher field strength. Field strength is inversely proportional to conductivity,
which is proportional to concentration.
• The result is that the three species of interest adjust their concentrations
so that [Cl-] > [protein–SDS] > [glycinate]. There is only a small quantity
of protein–SDS complexes, so they concentrate in a very tight band
between glycinate and Cl- boundaries. Once the glycinate reaches the
separating gel it becomes more fully ionized in the higher pH
environment and its mobility increases.(The pH of the stacking gel is 6.8,
that of the separating gel is 8.8.) Thus, the interface between glycinate
and Cl- leaves behind the protein–SDS complexes, which are left to
electrophorese at their own rates.
• The negatively charged protein–SDS complexes now continue to move
towards the anode, and, because they have the same charge per unit
length, they travel into the separating gel under the applied electric field
separate same mobility.
• However, as they pass through the separating gel the proteins separate,
owing to the molecular sieving properties of the gel.
• Quite simply, the smaller the protein the more easily it can pass
through the pores of the gel, whereas large proteins are successively
retarded by frictional resistance due to the sieving effect of the gels.
• Being a small molecule, the bromophenol blue dye is totally
unrewarded and therefore indicates the electrophoresis front.
• When the dye reaches the bottom of the gel, the current is turned off,
and the gel is removed from between the glass plates and shaken in
an appropriate stain solution (usually Coomassie Brilliant Blue, and
then washed in distain solution.
• The distain solution removes unbound back-ground dye from the gel,
leaving stained proteins visible as blue bands on a clear background
• A typical mingle would take about 1 h to prepare and set, 40 min to
runt 200 V and have a 1 h staining time with Coomassie Brilliant Blue.
• Upon distaining, strong protein bands would be seen in the gel within
10-20 min, but overnight distaining is needed to completely remove
all background stain. Vertical slab gels are invariably run, since this
allows up to 10 different samples to be loaded onto a single gel.
The following table shows the distance moved in an SDS–
polyacrylamide gel by a series of marker proteins of known relative
molecular mass (Mr.). A newly purified protein (X) run on the same
gel showed a single band that had moved a distance of 45 mm
• Construct a calibration graph by plotting log Mr. versus distance
moved for each of the marker proteins. From a graph of log Mr. versus
the distance moved by each protein you can determine a relative
molecular mass for protein X
Native buffer
• Problem- enzyme activity cannot be determined as the nature of
protein is destroyed by SDS hence native buffer are used . Usually do
not contain any SDS. The separation is carried on the basis of
molecular size and each sample carries their native charge at pH of
gel
• The gel is then recovered and substrate are added further locating
agent is added to locate enzyme. the diffusion and interaction of
enzyme and substrate between the two gels results in color formation
at the site of the enzyme
Gradient buffer
• Instead of uniform pore size (15%) gel slab, gradient pore size gel slab
used (5-25%).
• Therefore at the top of the gel there is a large pore size (5% acrylamide)
but as the sample moves down through the gel the acrylamide
concentration slowly increases and the pore size correspondingly
decreases. Gradient gels are normally run as SDS gels with a stacking gel.
• ADVANTAGE
• 1) much greater range of protein Mr. values can be separated than on a
fixed-percentage gel. In a complex mixture, very low molecular weight
proteins travel freely through the gel to begin with, and start to resolve
when they reach the smaller pore sizes towards the lower part of the gel.
Much larger proteins, on the other hand, can still enter the gel but start
to separate immediately due to the sieving effect of the gel.
• 2)proteins with very similar Mr. values may be resolved, although they
cannot otherwise be resolved in fixed percentage gels. As each
protein moves through the gel the pore sizes become smaller until the
protein reaches its pore size limit. The pore size in the gel is now too
small to allow passage of the protein, and the protein sample stacks
up at this point as a sharp band. A similar-sized protein but with
slightly lower Mr. will be able to travel a little further through the gel
before reaching its pore size limit, at which point it will form a sharp
band. These two proteins, of slightly different Mr. values, therefore
separate as two, close, sharp bands.
ISOELECTRIC ELECTROPHORESIS
• This method is highly efficient because separation is based on their
ISOELECTRIC point (PI).
• Minor diff 0.01PH can also be detected by this method and can be
separated.
• Relatively higher cost but can be made economical by using thinner
gel sheets
• Also we do not separation on mol wt. basis so gel sheet must not
cause any frictional resistance or hindrance separation should be on
basis of PI only.
• PREPARATION OF GEL(IEF GEL)
• Material needed :
• 1.Carrier ampholyte.
• 2.Acrylamide soln.
• 3.Riboflavin

• Take a glass plate containing space . Mix above mentioned ingredients

and fill them in space slowly . Again cover them with another glass
plate . Expose in direct sunlight or radiation to generate free radicals.
• These free radicals were generated by thermal degradation of
riboflavin
• Production of free radicals initiate the polymerization reaction of
acrylamide molecules(4-5hrs).
• Once the gel has been set after polymerization, the glass plates are
separated apart to extract the gel layer and electrodes are inserted
• NOTE: The ampholytes like PHARMALYTE and BIOLYTE are
commercially available and posses variable pH ranges. They are
mainly responsible for producing pH gradient across the gel.
• PROCEDURE:
• Let’s say with example , amino acids are taken as sample whose PI are
less than the pH of gel media . Initially the amino acids were in
amphoteric state , but due to gel higher pH they release H+ ions and
gets anionic state , because of acquiring negative charge they will
move towards the positive electrode which is anode (lower )
• The amino acids whose PI was greater than the pH of gel sheet will
acquire +vee charge by releasing OH- ions. And move towards
cathode which is –very charged.
• Now , as the amino acids move across the gel , they experience the
pH change because we have used gradient pH acrylamide gel with
ampholytes .
• Further , At a point when pH of gel becomes exactly equal to PI of
amino acid , the amino acid will get neutrally charged hence further
movement will be stopped.
• When the separation is complete, the current is switched off and the
sample components run out through a valve in the base of the
column.
This shows typical ISOELECTRIC focusing
electrophoresis . Track 1 contains mixture
Of std proteins with known PI.
[2-D] SDS PAGE electrophoresis
• Consist of 2 types of gel.
• First is ISOELECTRIC focusing gel layer which separates the sample on
basis of their PI
• Second layer is SDS-PAGE which separates the sample on basis of
molar mass
• This method is generally used for separation of complex proteins
• PROCEDURE: First take isoelectric gel and apply the sample of
complex proteins on that.
• Apply the current to both electrodes attached to gel.
• Wait till the proteins gets separated on basis of PI.
• Now it’s turn for the SDS – PAGE electrophoresis. For this rotate the
previous gel at 90° and attach it SDS gel.
• This gel contains sodium dodecyl sulphate which denatures the
protein sample by breaking it’s disulfide bond, B- mercaptothion
which breaks H bond And lastly bromophenol blue which acts as
visualizing agent.
• When we apply the electric field , the molecule further separate on
basis of their molecular weight.
Reference:
• Principles and techniques of biochemistry and molecular biology 7th
edition by Keith Wilson and John Walker, page no: 420-422