Electrophoresis

Acrylamide gels
• Electrophoresis in acrylamide gels is frequently referred to as PAGE, being an abbreviation for
polyacrylamide gel electrophoresis.

• Cross-linked polyacrylamide gels are formed from the polymerisation of acrylamide monomer in the
presence of smaller amounts of N,N’-methylene-bisacrylamide (normally referred to as ‘bis’-
acrylamide)

• Acrylamide monomer is polymerised in a head-to-tail fashion into long chains and occasionally a bis-
acrylamide molecule is built into the growing chain, thus introducing a second site for chain extension.

• Proceeding in this way a cross-linked matrix of fairly well-defined structure is formed.

• The polymerisation of acrylamide is an example of free-radical catalysis, and is initiated by the addition
of ammonium persulphate and the base N,N,N’,N’-tetramethylenediamine (TEMED).

• TEMED catalyses the decomposition of the persulphate ion to give a free radical (i.e. a molecule with
an unpaired electron):
• Free radicals are highly reactive species due to the presence of an unpaired
electron that needs to be paired with another electron to stabilise the
molecule.

• The free radical (R) therefore reacts with a monomer molecule (M),
forming a single bond by sharing its unpaired electron with one from the
outer shell of the monomer molecule. This produces a new free radical
molecule RM*, which is equally reactive and will attack a further monomer
molecule.

• In this way long chains of acrylamide are built up, being cross-linked by the
introduction of the occasional bis-acrylamide molecule into the growing
chain.

• Oxygen mops up free radicals and therefore all gel solutions are normally
degassed (the solutions are briefly placed under vacuum to remove loosely
• Acrylamide gels are defined in terms of the total percentage of
acrylamide present, and the pore size in the gel can be varied by
changing the concentrations of both the acrylamide and bis-
acrylamide.

• Acrylamide gels can be made with a content of between 3% and 30%

acrylamide. Thus low percentage gels (e.g. 4%) have large pore sizes
and are used, for example, in the electrophoresis of proteins as the
stacking gel.

• Gels of between 10% and 20% acrylamide are used in techniques such
as SDS–gel electrophoresis, where the smaller pore size now
introduces a sieving effect that contributes to the separation of
proteins according to their size.
 SDS-PAGE
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 Sample Loading Buffer
• SDS (CH3-(CH2)10 -CH2OSO3- Na+ ) is an anionic detergent. Samples to be run on SDS–PAGE are firstly
boiled for 5 min in sample buffer containing ß-mercaptoethanol and SDS. The mercaptoethanol
reduces any disulphide bridges present that are holding together the protein tertiary structure, and
the SDS binds strongly to, and denatures, the protein.
• 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 ionisable tracking dye, usually bromophenol blue, that allows
the electrophoretic 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.
 Stacking Gel and Band Sharpening
• 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 utilising differences in ionic strength and pH between the electrophoresis
buffer and the stacking gel buffer and involves a phenomenon known as isotachophoresis.

• 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 than 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 ionised 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 with the same mobility.
• SDS-polyacrylamide gel electrophoresis (SDS-PAGE) separates proteins on the basis of their shape
(size), which in turn relates to their relative molecular masses.

• A series of proteins of known molecular mass (molecular weight markers) are run on a gel on a track
adjacent to the protein of unknown molecular mass.

• The distance each marker protein moves through the gel is measured and a calibration curve of log
Mr versus distance moved is plotted.

• The distance migrated by the protein of unknown Mr is also measured, and from the graph its log Mr
and hence Mr is calculated.

• The method is suitable for proteins covering a large Mr range (10 000–300 000). Gels of 15%
polyacrylamide are useful for separating proteins in the range Mr 100 000 to 10 000.

• The method is easy to perform and requires very little material. If silver staining is used, as little as 1
ng of protein is required.
NATIVE PAGE
 Principle
• While SDS–PAGE is the most frequently used gel system for studying proteins, the method is of no use if
one is aiming to detect a particular protein (often an enzyme) on the basis of its biological activity,
because the protein (enzyme) is denatured by the SDS–PAGE procedure.

• In this case it is necessary to use non-denaturing conditions. In native or buffer gels, polyacrylamide gels
are again used (normally a 7.5% gel) but the SDS is absent and the proteins are not denatured prior to
loading.

• Since all the proteins in the sample being analysed carry their native charge at the pH of the gel (normally
pH 8.7), proteins separate according to their different electrophoretic mobilities and the sieving effects of
the gel.

• It is therefore not possible to predict the behaviour of a given protein in a buffer gel but, because of the
range of different charges and sizes of proteins in a given protein mixture, good resolution is achieved.

• The enzyme of interest can be identified by incubating the gel in an appropriate substrate solution such
that a coloured product is produced at the site of the enzyme. An alternative method for enzyme
detection is to include the substrate in an agarose gel that is poured over the acrylamide gel and allowed
to set. Diffusion and interaction of enzyme and substrate between the two gels results in colour formation
at the site of the enzyme.
 Gradient gels
• This is again a polyacrylamide gel system, but instead of running a slab gel of
uniform pore size throughout (e.g. a 15% gel) a gradient gel is formed, where
the acrylamide concentration varies uniformly from, typically, 5% at the top
of the gel to 25% acrylamide at the bottom of the gel. The gradient is formed
via a gradient mixer and run down between the glass plates of a slab gel.

• There are two advantages to running gradient gels.

• First, a 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.

• The second advantage of gradient gels is that proteins with very similar Mr values
may be resolved, although they cannot otherwise be resolved in fixed percentage
gels.
 Iso-electric Focusing
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 Principle
• Separation is achieved by applying a potential difference across a gel that contains a pH gradient.

• The pH gradient is formed by the introduction into the gel of compounds known as ampholytes, which are complex
mixtures of synthetic polyaminopolycarboxylic acids.

• Ampholytes can be purchased in different pH ranges covering either a wide band (e.g. pH 3-10) or various narrow bands
(e.g. pH 7-8), and a pH range is chosen such that the samples being separated will have their isoelectric points (pI
values) within this range.

• Since this method requires the proteins to move freely according to their charge under the electric field, IEF is carried
out in low percentage gels to avoid any sieving effect within the gel.

• Depending on which point on the pH gradient the sample has been loaded, proteins that are initially at a pH region
below their isoelectric point will be positively charged and will initially migrate towards the cathode. As they proceed,
however, the surrounding pH will be steadily increasing, and therefore the positive charge on the protein will decrease
correspondingly until eventually the protein arrives at a point where the pH is equal to its isoelectric point. The protein
will now be in the zwitterion form with no net charge, so further movement will cease.

• Likewise, substances that are initially at pH regions above their isoelectric points will be negatively charged and will
migrate towards the anode until they reach their isoelectric points and become stationary.
 Calculating pI of a Protein
• The pI of a particular protein may be determined conveniently by
running a mixture of proteins of known isoelectric point on the same
gel. A number of mixtures of proteins with differing pI values are
commercially available, covering the pH range 3.5-10.

• After staining, the distance of each band from one electrode is

measured and a graph of distance for each protein against its pI
(effectively the pH at that point) plotted.

• By means of this calibration line, the pI of an unknown protein can be

determined from its position on the gel.
2D Gel Electrophoresis
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 Introduction
• 2-D PAGE has found extensive use in detecting changes in gene expressions
between two different biological states, for example comparing normal
and diseased tissue.
• In this case, a 2-D gel pattern would be produced of an extract from a
diseased tissue such as a liver tumour and compared with the 2-D gel
patterns of an extract from normal liver tissue.
• The two gel patterns are then compared to see whether there are any
differences in the two patterns.
• If it is found that a protein is present (or is absent) only in the liver tumour
sample, then by identifying this protein we are directed to the gene for
this protein and can thus try to understand why this gene is expressed (or
not) in the diseased state.
• In this way it is possible to obtain an understanding of the molecular basis
of diseases.
 Advantages of 2-D
Electrophoresis
• Tolerant to crude sample loads: no prepurification (like
chromatography) has to be employed.
• Extremely high resolution.
• 2-D gels are a very effective fraction collectors
• Proteins are protected inside the gel matrix
 Cell disruption methods
• Freeze-thaw or osmotic lysis
• Detergent lysis
• Sonication
• Enzymatic lysis
• French pressure cell
• Grinding (mortar and pestle)
• Mechanical homogenization
 Protein precipitation
• Ammonium sulfate • Not efficient, de-salting
(salting out) necessary

• TCA precipitation • Can be hard to re-solubilize

• Acetone and/or ethanol • Leaves SDS behind, but many

proteins not precipitated

• TCA plus acetone • More effective than either

alone, good for basic proteins
 First Dimension: Denaturing IEF
• High molar (8 mol/L) urea, thiourea
• one conformation of a protein
• for protein solubility
• prevents protein aggregates and hydrophobic interactions

• Non-ionic or zwitterionic detergent

• for protein solubility

• IPG Buffer (carrier ampholyte mixture)

• for protein solubility
• raises the conductivity of the DryStrips

• DTT, DTE (no 2-mercaptoethanol)

• prevents different oxidation steps
 Why Denaturing IEF?

IEF in presence of 8 - 9 mol/L urea, nonionic or zwitterionic detergent,

reductant:
• Proteins in one conformation
• Urea and detergent
• avoid protein-protein aggregats
• keep hydrophobic proteins in solution
• Different oxidation steps avoided
• Theoretical pIs match with denaturing pIs
 Immobiline DryStrips: 1 st

Generation

11 cm strips:
• pH 4 - 7
• pH 3 - 10
• pH 6 - 11
7 cm, 13 cm, 18 cm,
and 24 cm strips:
• pH 4 - 7
• pH 3 - 10 L (linear gradient)
• pH 3 - 10 NL (non-linear gradient)
• pH 6 - 11
Second Dimension on Vertical
Equipment
applying the
pipetting the IPG strip
agarose
overlay low
melting
agarose
 Effect of sample prep
technique
(Drosophila larva extract)
Homogenate precipitated with
Homogenized in 8 M urea, Homogenized in 2% SDS 80% acetone, 10% TCA.
4% CHAPS Heated at 95 ºC 3 min Resuspended in 8 M urea, 4% CHAPS

First dimension is pH 3-10 L run on IPGphor in 8 M urea, 2% CHAPS, 0.5% IPG buffer, 65 mM DTT
• Under favourable circumstances up to 5000 protein spots can be
identified on a large format 2-D gel. Thus with 2-D PAGE we now have
the ability to follow changes in the expression of a significant
proportion of the proteins in a cell or tissue type, rather than just one
or two, which has been the situation in the past.
• The potential applications of proteome analysis are vast:
• comparing normal tissue with diseased tissue;
• analyse the effects of drug treatment or toxins on cells;
• observe the changing protein component of the cell at different stages of
tissue development;
• observe the response to extracellular stimuli such as hormones or cytokines;
• compare pathogenic and non-pathogenic bacterial strains;
• compare serum protein profiles from healthy individuals and Alzheimer or
cancer patients to detect proteins, produced in the serum of patients, which
can then be developed as diagnostic markers for diseases
Comparison: E. Coli Protein Extract in IPG pH 6-11
7 cm 11 cm 13 cm 18 cm
Protein Detection Methods
Sensitivity Quantification Living cells Linear Dynamic
limit Range
Coomassie Blue 100 ng +++ no 3
staining
Negative staining 15 ng + no 3
Silver staining 200 pg ++ no 7
4
Fluorescent 400 pg ++++ no 10
staining
4
Fluorescent 250 pg ++ no 10
labelling
Radioactive
labeling:
X-ray film 1 pg +++ yes 20
5
Phospor-imager 0.2 pg ++++ yes 10
plates
Stable isotope < 1 pg ++++ yes ?
labelling (with MS)
 Coomassie Blue Stain
Characteristics
• Sensitivity down to 0.1 mg
• Non-specific protein stain
• Stoichiometric binding of dye to protein
• Good linearity
• Diffusion-limited, thickness of gel governs speed of process
• Inadequate purity (Mass Spec!?)
• “Colloidal” Coomassie procedures are the most sensitive
 Coomassie Blue Stain Method
• The most commonly used general protein stain for detecting protein on gels is the
sulphated trimethylamine dye Coomassie Brilliant Blue R-250 (CBB).
• Staining is usually carried out using 0.1% (w/v) CBB in methanol:water:glacial acetic acid
(45:45:10, by vol.).
• This acid–methanol mixture acts as a denaturant to precipitate or fix the protein in the
gel, which prevents the protein from being washed out whilst it is being stained.
• Staining of most gels is accomplished in about 2 h and destaining, usually overnight, is
achieved by gentle agitation in the same acid–methanol solution but in the absence of
the dye.
• The Coomassie stain is highly sensitive; a very weakly staining band on a polyacrylamide
gel would correspond to about 0.1 mg (100 ng) of protein.
• The CBB stain is not used for staining cellulose acetate (or indeed protein blots) because
it binds quite strongly to the paper.
• In this case, proteins are first denatured by brief immersion of the strip in 10% (v/v)
trichloroacetic acid, and then immersed in a solution of a dye that does not stain the
support material, for example Procion blue, Amido black or Procion S.
 Acid Violet 17 Staining:
(Patestos NP et al. Electrophoresis. 9 (1988) 488-496)

• fix for 20 min in 20% TCA,

• wash for 1 min in 3% phosphoric acid,
• stain for 10 min in 0.1 % Acid Violet 17 solution in 10% phosphoric acid,
• destain 3 ´ in 3% phosphoric acid until background is clear,
• wash 3 ´ 1 min with H2Odist,
• impregnate with 5 % glycerol,
• air dry.
 Imidazole-Zinc Reverse Stain
• Sensitivity down to 15 ng
• Clear spots on an opaque white background
• Compatible with subsequent blotting
• Quantification not very reliable (negative stain)
 Silver Stain Characteristics
• Sensitivity down to 0.2 ng for protein
• Stains a variety of macromolecules
• Stains primarily near the surface of the gel
• Autocatalytic reaction
• Less linear than Coomassie Blue staining
• Crosslinking of proteins into gel matrix
Silver nitrate vs Silver diamine
• Silver weakly bound • Silver strongly bound
• Relatively benign solution • Caustic solution
• Develop at high pH • 10 ´ silver conc.
• Can be modified to become MS- • Develop at low pH
compatible • More sensitive for basic proteins
 Silver Stain Method
• Although the Coomassie stain is highly sensitive, many workers require greater
sensitivity such as that provided by silver staining.
• Silver stains are based either on techniques developed for histology or on methods
based on the photographic process.
• In either case, silver ions (Ag+) are reduced to metallic silver on the protein, where
the silver is deposited to give a black or brown band.
• Silver stains can be used immediately after electrophoresis, or, alternatively, after
staining with CBB.
• With the latter approach, the major bands on the gel can be identified with CBB and
then minor bands, not detected with CBB, resolved using the silver stain.
• The silver stain is at least 100 times more sensitive than CBB, detecting proteins
down to 1 ng amounts.
• However, silver stains may interfere with Mass Spectrometry.
• Other stains with similar sensitivity include the fluorescent stains Sypro Orange (30
ng) and Sypro Ruby (10 ng).
Identification of differentially regulated
• The sheer complexity andproteins
amount of data available from 2-D gel
patterns is daunting, but fortunately there is a range of commercial 2-D
gel analysis software, compatible with personal computer workstations,
which can provide both qualitative and quantitative information from
gel patterns, and can also compare patterns between two different 2-D
gels.
• This has led to the construction of a range of databases of quantitative
protein expression in a range of tissue and cell types e.g. an extensive
series of 2-DE databases, known as SWISS-2D PAGE, is maintained at
Geneva University Hospital and is accessible via the World Wide Web (
http://au.expasy.org/ch2d/).
• The gel comparisons can then identify protein spots that occur either in
the normal state or in the diseased state only or protein spots that are
up-regulated or down-regulated in the diseased state.
 Identification of Proteins in 2D gels
• When one has compared two 2-DE patterns and identified any proteins spot(s) of
interest, it is then necessary to identify each specific protein.
• In the majority of cases this is done by peptide mass-fingerprinting.
• The spot of interest is cut out of the gel and incubated in a solution of the proteolytic
enzyme trypsin, which cleaves the protein C-terminal to each arginine and lysine
residue. In this way the protein is reduced to a set of peptides.
• This collection of peptides is then analysed by MALDI-MS to give an accurate mass
measurement for each of the peptides in the sample. This set of masses, derived from
the tryptic digestion of the protein, is highly diagnostic for this protein, as no other
protein would give the same set of peptide masses (fingerprint).
• Using Web-based programs such as Mascot or Protein Prospector this experimentally
derived peptide mass-fingerprint is compared with databases of tryptic peptide mass-
fingerprints generated from sequences of known proteins (or predicted sequences
deduced from nucleotide sequences).
• If a match is found with a fingerprint from the database then the protein will be
identified.
• However, sometimes results from peptide mass-fingerprinting can be ambiguous.
 Western Blots
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 Principle/Method
• The first step is to transfer or blot the pattern of separated proteins from
the gel onto a sheet of nitrocellulose paper. The method is known as
protein blotting, or western blotting by analogy with Southern blotting the
equivalent method used to recover DNA samples from an agarose gel.
• Transfer of the proteins from the gel to nitrocellulose is achieved by a
technique known as electroblotting. In this method a sandwich of gel and
nitrocellulose is compressed in a cassette and immersed, in buffer, between
two parallel electrodes.
• A current is passed at right angles to the gel, which causes the separated
proteins to electrophorese out of the gel and into the nitrocellulose sheet.
• The nitrocellulose with its transferred protein is referred to as a blot.
• Once transferred onto nitrocellulose, the separated proteins can be
examined further. This involves probing the blot, usually using an antibody
to detect a specific protein
 Detection
• The blot is first incubated in a protein solution, for example 10% (w/v) bovine
serum albumin, or 5% (w/v) non-fat dried milk, which will block all remaining
hydrophobic binding sites on the nitrocellulose sheet.
• The blot is then incubated in a dilution of an antiserum (primary antibody)
directed against the protein of interest.
• This IgG molecule will bind to the blot if it detects its antigen, thus identifying the
protein of interest.
• In order to visualise this interaction the blot is incubated further in a solution of a
secondary antibody, which is directed against the IgG of the species that provided
the primary antibody. For example, if the primary antibody was raised in a rabbit
then the secondary antibody would be anti-rabbit IgG.
• This secondary antibody is appropriately labelled so that the interaction of the
secondary antibody with the primary antibody can be visualised on the blot.
• Anti-species IgG molecules are readily available commercially, with a choice of
different labels attached.
 Antibody Labels in Western Blots
• One of the most common detection methods is to use an enzyme-
linked secondary antibody.
• Following treatment with enzyme-labelled secondary antibody, the
blot is incubated in enzyme–substrate solution, when the enzyme
converts the substrate into an insoluble coloured product that is
precipitated onto the nitrocellulose.
• The presence of a coloured band therefore indicates the position of
the protein of interest.
• The enzyme used in enzyme-linked antibodies is usually either:
• alkaline phosphatase, which converts colourless 5-bromo-4-chloro-
indolylphosphate (BCIP) substrate into a blue product, or
• horseradish peroxidase, which, with H2O2 as a substrate, oxidises either 3-
amino-9-ethylcarbazole into an insoluble brown product, or 4-chloro-
lnaphthol into an insoluble blue product.
Enhanced chemiluminescence (ECL)
• In the presence of hydrogen peroxide and the chemiluminescent
substrate luminol horseradish peroxidase oxidises the luminol with
concomitant production of light, the intensity of which is increased
1000-fold by the presence of a chemical enhancer.
• The light emission can be detected by exposing the blot to a
photographic film.
• Corresponding ECL substrates are available for use with alkaline-
phosphatase labelled antibodies.
• The principle behind the use of enzyme-linked antibodies to detect
antigens in blots is highly analogous to that used in enzyme-linked
immunosorbent assays (ELISA)
DNA Electrophoresis
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 Principle of DNA Gel Electrophoresis
• For the majority of DNA samples, electrophoretic separation is carried out in agarose
gels. This is because most DNA molecules and their fragments that are analysed routinely
are considerably larger than proteins and therefore, because most DNA fragments would
be unable to enter a polyacrylamide gel, the larger pore size of an agarose gel is required.
• Since the charge per unit length (owing to the phosphate groups) in any given fragment
of DNA is the same, all DNA samples should move towards the anode with the same
mobility under an applied electrical field.
• However, separation in agarose gels is achieved because of resistance to their movement
caused by the gel matrix. The largest molecules will have the most difficulty passing
through the gel pores (very large molecules may even be blocked completely), whereas
the smallest molecules will be relatively unhindered.
• Consequently the mobility of DNA molecules during gel electrophoresis will depend on
size, the smallest molecules moving fastest.
• While passing through the pores, a DNA molecule will experience drag; so the longer the
molecule, the more it will be retarded by each pore.
• Sideways movement may become more important for very small double-stranded DNA
and for the more flexible single-stranded DNA.
 Percentage of Agarose Gel
• Gel concentrations must be chosen to suit the size range of the
molecules to be separated.

• Gels containing 0.3% agarose will separate double-stranded DNA

molecules of between 5 and 60 kb size, whereas

• 2% gels are used for samples of between 0.1 and 3 kb.

• Many laboratories routinely use 0.8% gels, which are suitable for
separating DNA molecules in the range 0.5 - 10 kb.
 Determination of Size
• Since agarose gels separate DNA according to size, the Mr of a DNA
fragment may be determined from its electrophoretic mobility by
running a number of standard DNA markers of known Mr on the same
gel (DNA Ladders).

• This is most conveniently achieved by running a sample of

bacteriophage λ DNA (49 kb) that has been cleaved with a restriction
enzyme such as EcoRI.

• Since the base sequence of λ DNA is known, and the cleavage sites for
EcoRI are known, this generates fragments of accurately known size
 Buffers Used
• Buffers in gel electrophoresis are used to provide ions that carry a
current and to maintain the pH at a relatively constant value.

• These buffers have plenty of ions in them, which is necessary for the
passage of electricity through them.

• There are a number of buffers used for electrophoresis. The most

common for nucleic acids being:
• TBE (Tris/Borate/EDTA)
• TAE (Tris/Acetate/EDTA)
 TBE Buffer
• TBE or Tris/Borate/EDTA, is a buffer solution containing a mixture of Tris base,
boric acid and EDTA.

• In molecular biology, TBE and TAE buffers are often used in procedures involving
nucleic acids, the most common being electrophoresis.

• Tris-acid solutions are effective buffers for slightly basic conditions, which keep
DNA deprotonated and soluble in water.

• EDTA is a chelator of divalent cations, particularly of magnesium (Mg 2+). As these

ions are necessary co-factors for many enzymes, including contaminant
nucleases, the role of the EDTA is to protect the nucleic acids against enzymatic
degradation. But since Mg2+ is also a co-factor for many useful DNA-modifying
enzymes such as restriction enzymes and DNA polymerases, its concentration in
TBE or TAE buffers is generally kept low (typically at around 1 mM).
 TBE Recipe
• Recipe (1 liter of 5X stock solution)
• 54 g of Tris base
• 27.5 g of boric acid
• 20 ml of 0.5 M EDTA (pH 8.0)
• Adjust pH to 8.3 by HCl.

• TBE can be diluted to 1X prior to use in electrophoresis, 0.5x is

acceptable as well. Higher concentrations will result in poor results
due to excessive heat generation.
 TAE Buffer
• TAE buffer is a buffer solution containing a mixture of Tris base, acetic acid
and EDTA.
• In molecular biology it is used in agarose electrophoresis typically for the
separation of nucleic acids such as DNA and RNA.
• It is made up of Tris-acetate buffer, usually at pH 8.3, and EDTA, which
sequesters divalent cations.
• TAE has a lower buffer capacity than TBE and can easily become exhausted,
but linear, double stranded DNA runs faster in TAE.
• Compared with TBE buffer, TAE buffer offers advantages in subsequent
enzymatic applications for the DNA sample. For example, if a DNA sample is
going to be used in a cloning experiment, the step that follows its running
on an agarose gel is to ligate (covalently link) to a cloning vector (most likely
a plasmid). A DNA sample from a TAE gel is suitable for this purpose, while
DNA from a TBE gel is not, because borate in the TBE buffer is a strong
inhibitor for many enzymes
 TAE Recipe
• TAE buffer is commonly prepared as a 50X stock solution for
laboratory use.

• A 50X stock solution can be prepared by dissolving:

• 242g Tris base in water,
• adding 57.1mL glacial acetic acid, and
• 100mL of 500mM EDTA (pH 8.0) solution, and
• bringing the final volume up to 1 liter.

• This stock solution can be diluted 50:1 with water to make a 1X

working solution. This 1X solution will contain 40mM Tris, 20mM
acetic acid, and 1mM EDTA.
 Detection
• Once the system has been run, the DNA in the gel needs to be stained and visualised.
• The reagent most widely used is the fluorescent dye ethidium bromide.
• The gel is rinsed gently in a solution of ethidium bromide (0.5 mg cm3) and then
viewed under ultraviolet light (300nm wavelength).
• Ethidium bromide is a cyclic planar molecule that binds between the stacked base-
pairs of DNA (i.e. it intercalates).
• The ethidium bromide concentration therefore builds up at the site of the DNA
bands and under ultraviolet light the DNA bands fluoresce orange-red. As little as 10
ng of DNA can be visualised as a 1 cm wide band.
• It should be noted that extensive viewing of the DNA with ultraviolet light can result
in damage of the DNA by nicking and base-pair dimerisation. This is of no
consequence if a gel is only to be viewed, but obviously viewing of the gel should be
kept to a minimum if the DNA is to be recovered.
• It is essential to protect one’s eyes by wearing goggles when ultraviolet light is used.
If viewing of gels under ultraviolet is carried out for long periods, a plastic mask that
covers the whole face should be used to avoid ‘sunburn’.
 Capillary
Electrophoresis
https://www.youtube.com/watch?v=102gXzAVJ-8
https://www.youtube.com/watch?v=-rDRU_qxwYA
https://www.youtube.com/watch?v=wStV1rFjHOo
CE Instrument
 Introduction
• The microscale nature of the capillary used, where only microliters of reagent
are consumed by analysis and only nanolitres of sample needed for analysis,
together with the ability for on-line detection down to femtomole (10-15 moles)
sensitivity in some cases has for many years made capillary electrophoresis the
method of choice for many biomedical and clinical analyses.
• Capillary electrophoresis can be used to separate a wide spectrum of biological
molecules including amino acids, peptides, proteins, DNA fragments (e.g.
synthetic oligonucleotides) and nucleic acids, as well as any number of small
organic molecules such as drugs or even metal ions
• Capillary electrophoresis involves electrophoresis of samples in very narrow-
bore tubes (typically 50 µm internal diameter) which is coated with
Silanol/Silica.
• One advantage of using capillaries is that they reduce problems resulting from
heating effects.
• Voltages of 1050 kV with capillaries of 50-100 cm in length are commonly used.
 Characteristics
• Electrophoresis is performed in narrow-bore (25- to 75-!m id), fused silica
capillaries
• High voltages (10 to 30 kV) and high electric fields (100 to 500 V/cm) are
applied across the capillary
• High resistance of the capillary limits current generation and internal heating
• High efficiency (N>105 to 106) and short analysis time
• Detection performed on-capillary (no external detection cell)
• Small sample volume required (1 to 50 nl injected)
• Numerous modes to vary selectivity and wide application range
• Operates in aqueous media
• Simple methods development
• Automated instrumentation
 Sample Separation
• Electrophoretic migration causes the movement of charged molecules in
solution towards an electrode of opposite charge. Owing to this electrophoretic
migration, positive and negative sample molecules migrate at different rates.
• However, although analytes are separated by electrophoretic migration, they
are all drawn towards the cathode by electroendosmosis. Since this flow is
quite strong, the rate of electroendosmotic flow usually being much greater
than the electrophoretic velocity of the analytes, all ions, regardless of charge
sign, and neutral species are carried towards the cathode.
• Positively charged molecules reach the cathode first because the combination
of electrophoretic migration and electroosmotic flow causes them to move
fastest.
• As the separated molecules approach the cathode, they pass through a viewing
window where they are detected by an ultraviolet monitor that transmits a
signal to a recorder, integrator or computer. Typical run times are between 10
and 30 min.
 Electroendosmosis (electroosmotic flow)
• Due to the presence of charged groups on the
surface of the support medium
• In a fused-silica capillary tube, above a pH value
of about 3, silanol groups on the silica capillary
wall will ionise, generating negatively charged
sites.
• It is these charges that generate
electroendosmosis.
• The ionised silanol groups create an electrical
double layer, or region of charge separation, at
the capillary wall/electrolyte interface.
• When a voltage is applied, cations in the
electrolyte near the capillary wall migrate
towards the cathode, pulling electrolyte solution
with them.
• This creates a net electroosmotic flow towards
the cathode.
 Sample Application
Sample application is carried out in one of two ways, by high voltage injection or by pressure
injection:
• High voltage injection.
• With the high voltage switched off, the buffer reservoir at the positive electrode is replaced by a reservoir
containing the sample, and a plug of sample (e.g. 530 mm3 of a 1 mg cm3 solution) is introduced into the
capillary by briefly applying high voltage.
• The sample reservoir is then removed, the buffer reservoir replaced, voltage again applied and the
separation is then commenced.
• Pressure injection.
• The capillary is removed from the anodic buffer reservoir and inserted through an air-tight seal into the
sample solution. A second tube provides pressure to the sample solution, which forces the sample into the
capillary.
• The capillary is then removed, replaced in the anodic buffer and a voltage applied to initiate
electrophoresis.
• A high voltage (up to 50 kV) is then put across the capillary tube and component molecules in the injected
sample migrate at different rates along the length of the capillary tube.