Gel Electrophoresis

Advanced article

Reiner Westermeier, Serva Electrophoresis GmbH, Heidelberg, Germany

Article Contents
. Principle of Gel Electrophoresis
. Agarose Gel Electrophoresis
. Polyacrylamide Gel Electrophoresis

Online posting date: 15th February 2013

Gel electrophoresis is the core separation technique for

genetic analysis and purification of nucleic acid fragments for further studies. In an electric field the negatively charged deoxyribonucleic acid (DNA) and
ribonucleic acid (RNA) fragments migrate through a
porous gel matrix toward the positive electrode, the
anode. Because of the sieving effect of the gel, shorter
fragments move faster than larger ones. In this way the
DNA or RNA samples are separated according to their
molecular sizes into distinct zones, which can be detected
by specific visualisation methods. The most frequently
used technique is the separation of DNA fragments in
agarose gels in simple flatbed boxes combined with the
detection of stained bands under ultraviolet light. Polyacrylamide gels are employed, when small fragments have
to be analysed or very high resolution down to one single
base pair is required. In contrast to capillary electrophoresis the substrate does not need to be prelabelled.

Principle of Gel Electrophoresis

Electrophoresis is the migration of charged particles or
molecules in an electric eld. This occurs when the substances are in aqueous solution. The speed of migration is
dependent on the applied electric eld strength and the
charges of the molecules. Thus, dierently charged molecules will form individual zones while they migrate. To
keep diusion of the zones to a minimum, electrophoresis is
carried out in an anticonvective medium such as a viscous
uid or a gel matrix. Therefore, the speed of migration is
also dependent on the size of the molecules. In this way
fractionation of a mixture of substances is achieved with
high resolution.

Electrophoretic mobility
The electrophoretic mobility is dependent on external factors like electric eld strength, viscosity, gel concentration
eLS subject area: Molecular Biology
How to cite:
Westermeier, Reiner (February 2013) Gel Electrophoresis. In: eLS.
John Wiley & Sons, Ltd: Chichester.
DOI: 10.1002/9780470015902.a0005335.pub2

and temperature and intrinsic properties of the molecule like

charge density, size and hydrophobicity.
Although proteins can be separated according to their
net charges or their sizes, nucleic acid molecules are only
distinguishable on size-based separations in which the
properties of the separation medium have a large inuence
on the distribution of the zones.

Buffers
Electrophoretic separation is performed in buers with a
constant pH value and constant ionic strength. For nucleic
acid separation the buer must have a basic pH value to
ensure that the sample molecules are suciently charged.
During electrophoresis, the buer ions are carried through
the gel just like the sample ions: negatively charged
ions toward the anode, positively charged ones toward
the cathode. To guarantee constant pH and buer conditions, the supply of electrode buers must be sucient.
For nucleic acids the mostly used buer is composed of
tris(hydroxymethyl)-aminoethane (Tris), borate and
ethylenediaminetetraacetic acid (EDTA; TBE). These TBE
buers are used in concentrations from 45 to 90 mM Tris
borate and 1 to 2 mM EDTA and have a pH of 8.08.3.

Joule heat
Some of the electrical energy is transformed into Joule heat.
Development of Joule heat is increased with high buer
concentrations. To prevent overheating eects, buer
strength and electric eld strength must be limited and
mostly for polyacrylamide gels thermostating of the gels
provides a homogeneous temperature distribution. When
the conditions are not chosen correctly, a so-called smiling
eect will occur: the electrophoretic mobilities of ions are
higher in the hot centre of the gel plate than at the cooler
lateral sides.

Gel medium
The gel medium prevents diusion and thermal convection
of the zones, and serves as a molecular sieve. Two gel types
are employed: agarose and polyacrylamide gels. Agarose
gels are used as thick layers in atbed chambers mainly for
preparative purposes, whereas polyacrylamide gels are
applied in thin layers in vertical or cooled atbed systems,
mainly for high-resolution techniques like sequencing and
genotyping.

eLS & 2013, John Wiley & Sons, Ltd. www.els.net

Gel Electrophoresis

Electroendosmosis
The stabilising medium, particularly agarose, can contain
xed carboxylic and sulfonic groups. In the presence of basic
and neutral buers, these groups will become deprotonated
and thus negatively charged. In the electric eld, the xed
negative charges are attracted by the anode. They cannot
migrate, because they are a part of the matrix. A counterow
of hydrated protons H3O+ toward the cathode will result in
compensation; this eect is termed electroendosmosis. In
gels, electroendosmosis is observed as a ow of water toward
the cathode, which carries some of the solubilised substances
along. The electrophoretic and electro-osmotic migrations
are subtractive, which results in blurred zones. Drying of the
gel in the area of the anode can also occur.

(a)

Agarose Gel Electrophoresis

Buffer
(b)

Properties of agarose gels

Agarose is a polysaccharide obtained from red seaweed. The
pore size depends on the concentration of agarose (weight of
agarose per volume). Agarose is dissolved in boiling water
and forms a gel during cooling. During this process, double
helices are built, which are joined laterally to form relatively
thick laments. This fact allows the preparation of gels with
large pore sizes and high mechanical stability. Gels with a
pore size from 150 nm at 1% (w/v) to 500 nm at 0.16% are
used. This allows separation of nucleic acid fragment sizes in
the range between 400 and 23 000 base pairs (bp).
Dierent agarose qualities are available. They are characterised by their gelling temperature (down to 358C),
melting point (down to 608C) and the degree of electroendosmosis. The degree of electroendosmosis is dependent
on the number of polar groups remaining from agaropectin.
The 110 mm thick gels are cast by pouring the hot
agarose mixed with gel buer onto ultraviolet (UV)transparent trays. Sample application wells are formed in
the gel surface with inserted plastic combs during gelling
(Figure 1a). The gel sizes vary from 5 cm to approximately
25 cm separation distances.

Running conditions and properties

Electrophoresis setup
Agarose gels are run in simply designed atbed chambers
under a buer layer to prevent drying due to electroendosmosis (Figure 1b). The technique is therefore often
called submarine electrophoresis. The temperature is only
controlled by the applied running conditions. The nucleic
acids are separated under native conditions. Quick checks
of multiple samples are performed in 96-well agarose gels in
microtiter plate format without a buer layer.

Migration of deoxyribonucleic acid fragments

Because of the sieving properties of agarose gels, the relative mobilities of deoxyribonucleic acid (DNA) and
2

Figure 1 Schematic drawing of a chamber for agarose gel electrophoresis:

(a) casting tray with comb for forming sample wells and (b) chamber with
gel and buffer. (+) and (2) are the anodal and cathodal platinum electrode
wires. The samples are applied into the sample wells in the gel. The gel is
covered with running buffer.

ribonucleic acid (RNA) molecules are dependent on the

sizes of the molecules. At a dened pore size of the agarose
gel, there is within a certain molecule size range a linear
relationship between the logarithms of the fragment
lengths and the relative migration distances.
Under the inuence of the electric eld, nucleic acid
molecules are stretched and migrate through gel pores like
a snake with a reptating movement (Noolandie et al.,
1989). Above a certain molecule length of approximately
20 kbp, the electrophoretic mobilities of DNA molecules
are similar, because these long chains keep to the same
orientation. When the applied eld strength exceeds a
certain value, the DNA molecules are so strongly stretched
that they become rigid rods. This results in poor separation.

Staining of the bands

The bands are visualised with uorescent dyes that are
visible in UV light ethidium bromide or SYBR Green.
SYBR Green is less mutagenic and more sensitive than
ethidium bromide. The best results and highest resolutions
are obtained when the gels are stained after the run. When
dyes are added to the gel or the sample during electrophoresis, the mobilities of the DNA fragments will be
modied and the resolution will suer.
Between 100 pg and 1 ng per band are detected. The dyes
intercalate in the helix and stain proportionately to
the length of the molecule. Therefore the sensitivity is
dependent on the size of the DNA fragment, and is lower
for single-stranded DNA and RNA.
The new DNA Stain G is a safer alternative to ethidium
bromide and SYBR Green. It is as sensitive as ethidium
bromide and can be used in exactly the same way in agarose

eLS & 2013, John Wiley & Sons, Ltd. www.els.net

Gel Electrophoresis

Pulsed field gel electrophoresis

23 130 bp

2322 bp

1057 bp
612 bp
335 bp

Figure 2 Separation result of agarose gel electrophoresis. DNA fragments

are detected with ethidium bromide.

gel electrophoresis. When it gets attached to the phosphate

groups of the nucleic acids it emits green light. Its adsorption maximum is at 530 nm, however, it can be detected
with an UV illuminator.
However, it must be noted that open UV tables are
hazardous to health; great care must be taken to protect the
eyes and skin from contact with UV light. For a permanent
record of the separation, instant photos are taken on the
UV table or video documentation systems are employed.
Figure 2 shows ethidium bromide stained bands in an
agarose gel.

DNA fragments longer than approximately 20 kb cannot be

resolved in conventional agarose gel electrophoresis because
long DNA molecules align themselves as rods and migrate
with a mobility that is independent of their length. In pulsed
eld gel electrophoresis (PFGE), the molecules are subjected
to two alternating electrical elds that are applied on the
gel at an angle between 1108 and 1808. The DNA fragments
must change their orientation with changes in the electric
eld: their helical structure is rst compressed and then
stretched. The viscoelastic relaxation time is dependent on
the size of the molecule (Schwartz and Cantor, 1984). In
addition, large molecules need more time to change their
direction than small ones. Because of the longer time needed
for stretching and reorientation, larger molecules have less
time left for migration in the electric eld. In PFGE, the
resulting electrophoretic mobilities depend on the pulse
time: DNA molecules with fragment sizes up to approximately 10 Mb can be resolved.
Pulse times of 1 s to 90 min are applied, depending on the
length of the DNA molecules being analysed. Large molecules are better separated with long pulse times, small
molecules need short pulse times. Separations can take
several days.
To prevent chromosome-size molecules breaking by
shear forces during pipetting, sample preparation including cell disruption is carried out inside little agarose blocks.
These agarose blocks are inserted into preformed sample
wells of the separation gel.

PFGE at different angles

Blotting and hybridisation

For restriction fragment length polymorphism analysis,
the separated DNA fragments are transferred onto an
immobilising membrane followed by hybridisation with
radiolabelled probes (Southern, 1975). The molecules are
transferred onto nitrocellulose or nylon membranes with
capillary forces. The fragments are probed with radioactive
DNA or RNA. The bound complementary nucleic
acids are detected by autoradiography. See also: Nucleic
Acids: Hybridisation; Southern Blotting for the Analysis of
Human Disease

Recovery of DNA fragments from gels

Several dierent procedures are used for the isolation of
nucleic acids from agarose gels: electroelution, absorption
to Diethylaminoethyl (ion exchange) paper, absorption
to glass powder or resins and digestion of agarose with
enzymes. For preparative electrophoresis, it is very
important to use highly puried agarose that is free from
polymerase and other enzyme inhibitors. Since the advent
of polymerase chain reaction (PCR) technology, tiny
amounts of DNA fragments can easily be amplied for
further experiments. See also: Genomic DNA: Purication

The directions of the applied electric elds must dier at least

by an angle of 1108. This is achieved by dierent arrangements: inhomogeneous elds created with point electrodes,
hexagonal electrode sets, turning electrodes or turning gel
tables. The resulting migration direction is diagonal. Figure 3
shows the principle for two types of PFGE.

Field inversion gel electrophoresis

Field inversion gel electrophoresis is performed in a
standard agarose gel electrophoresis apparatus. The electric elds are just alternating in the direction of 1808. The
resulting migration in one direction is achieved by applying
a higher eld strength or longer pulse time in the separation
direction. The advantage of this method is the simple
design. The disadvantage is the long separation time,
because the molecules migrate backwards for part of the
time. A wide range of sizes of DNA molecules can be
resolved in such gels.

Applications of PFGE
The eld of application of this technique includes
chromosome mapping, isolation of intact chromosomal
and chromosomal-sized DNA, large restriction fragment
mapping and karyotyping. With PFGE, physical gene
maps are created for the identication of genes responsible

eLS & 2013, John Wiley & Sons, Ltd. www.els.net

Gel Electrophoresis

Contour homogeneous electric field

electrophoresis
+

Field inversion gel

electrophoresis

+
+

+
+

+
+

+
+

+
+

Figure 3 Schematic drawing of the principle of pulsed field gel

electrophoresis.

for hereditary diseases. Another important area of application is bacterial taxonomy.

Polyacrylamide Gel Electrophoresis

Properties of polyacrylamide gels
Polyacrylamide gels are prepared by chemical copolymerisation of acrylamide monomers with a cross-linking
reagent, usually N,N-methylenebisacrylamide. A clear
transparent gel is obtained, which is chemically inert,
mechanically stable and without electroendosmosis.
Polymerisation of the acrylamide monomers and the
cross-linker molecules occurs in the presence of free radicals. These are provided by ammonium persulfate as
catalyst; tertiary amino groups, usually N,N,N,N-tetramethylethylenediamine, are required as accelerators.
The pore size is exactly controlled with the total acrylamide concentration (T) and the degree of cross-linking (C),
which is determined by the amount of cross-linker relative
to the total amount of acrylamide. The pore size decreases
with increasing T value. With increasing cross-linking, the
pore size follows a parabolic function: at high and low
cross-linking, the pores are large and the minimum pore
size is obtained at 4% cross-linking. Sequencing gels contain 5% cross-linking and gels for single-strand conformation polymorphism analysis 2% cross-linking.
Acrylamide monomers are toxic and should be handled
with caution. Because oxygen is a scavenger of free radicals,
polymerisation is performed in closed cassettes. Sample
application wells for vertical gels are formed at the upper
edge of the gel during polymerisation with the help of an
inserted comb (Figure 4). Sample wells for atbed gels are
made by using self-adhesive tape glued onto one of the glass
plates.

Running conditions and properties

For electrophoresis in vertical systems, the complete gel
cassettes are placed into the buer tanks; the gels are in
4

Figure 4 Schematic drawing of a cassette with sample well comb and a

caster for polyacrylamide gels. For electrophoresis the cassette containing
the gel layer is removed from the caster and inserted into the
electrophoresis chamber (Figure 5).

direct contact with the electrode buers. Gels for atbed

systems are polymerised on a lm support and removed
from the cassette before use. Figure 5a shows an example
of a atbed and Figure 5b a vertical chamber for polyacrylamide gels.

Native conditions
In nondenaturing polyacrylamide gels, the mobility of
DNA fragments is dependent on both size and sequence. Aand T-rich nucleic acids migrate faster, because they
undergo fewer hydrophobic interactions with the gel matrix than C- and G-rich fragments. Therefore, nondenaturing polyacrylamide gels cannot be used for the
determination of fragment length, but they are very sensitive to conformation dierences of the secondary structure.
Very sharp bands are obtained (Figure 6). Single-nucleotide
polymorphisms and point mutations are detected with high
sensitivity.

Denaturing conditions
In the presence of high molar formamide or urea, and at
elevated temperature above 508C, the DNA molecules are
completely denatured and exist as single strands. In this
case, the electrophoretic mobilities are strictly size
dependent. When thin gel layers are used, the resolution

eLS & 2013, John Wiley & Sons, Ltd. www.els.net

Gel Electrophoresis

Electrodes

Electrode wicks

(a)

(b)

Electrode
buffer

Figure 5 Schematic drawing of chambers for polyacrylamide gel

electrophoresis: (a) Horizontal flatbed chamber with cooling plate: The gel
is used with an open surface, the samples are applied into the sample wells,
instead of electrode reservoirs disposable wicks are soaked in concentrated
buffer, the electrodes are placed onto these wicks and connected to a
power supply. (b) Vertical chamber using liquid buffer: The samples are
applied into the wells located between the two glass plates, which have
been formed by the comb shown in Figure 4, the upper buffer tank is
located in the central block and contains a cathodal platinum electrode
wire, the lower buffer tank at the bottom contains an anodal platinum
electrode wire, the contacts to the power supply are made via the two
plugs located at the top of the central block.

reaches single-base dierence within a range of approximately 10001200 bases, which makes DNA sequencing
possible. See also: Denaturing Gel Electrophoresis of RNA
and DNA Using UreaPolyacrylamide Gels

Detection of bands
Staining
Ethidium bromide and SYBR Green staining are rarely
used for polyacrylamide gels, because the signals are
weaker than in agarose gels.
With silver staining, very high sensitivity independent of
molecular size is reached, down to 15 pg per band (Goldman and Merril, 1982). The staining method requires several steps; staining automates are available. The chemicals

Figure 6 Separation result of polyacrylamide gel electrophoresis of DNA

fragments with silver staining. On the lane on the right edge 5 mL of a
100 bp ladder, diluted to 10 ng, has been applied.

are less toxic than intercalating dyes, there is no radioactivity, no UV light and no photography is needed for
inspection of the results. Silver-stained bands can be directly reamplied with PCR without any intermediate
purication step. See also: Gel Staining Techniques

Radioactive labelling
Labelling with radioactive phosphorus (32P) during transcription or replication is employed for various applications because of its very high sensitivity of detection.
After the run, the gels are dried and exposed on X-ray
lm. The major applications are sequencing, amplied
fragment length polymorphism, dierential display reverse
transcription and two-dimensional DNA typing. See also:
Radiolabelling Nucleic Acids: Generating DNA Probes by
Random Priming

Fluorescence labelling
Labelling of the DNA fragments with Cy5 and other uorophors has replaced radiolabelling for many applications.

eLS & 2013, John Wiley & Sons, Ltd. www.els.net

Gel Electrophoresis

It allows online detection of the migrating zones. The dyes

are excited with a laser beam and the emitted light with a
dierent wavelength is measured with a diode detector.

DNA sequencing gels

For increasing the reading length, long gels in very thin
layers are optimal. To achieve a straight front and straight
band distribution over the entire gel width, the gels are
mostly heated with thermoplates. Fluorescent labelling has
generally replaced radiolabelling, which makes the long
ultrathin layer gels (Sanger and Coulson, 1978) and wedge
gels unnecessary (Ansorge and Labeit, 1984). DNA
sequencing in gels has meanwhile more or less been abandoned: rst it had been replaced by capillary electrophoresis in multicapillary systems, then by faster and
cheaper gel-free next generation sequencing methods.
See also: Capillary Electrophoresis; Capillary Electrophoresis; Sanger, Frederick

Denaturing gradient gel electrophoresis

Denaturing gradient gel electrophoresis (DGGE) aords
the detection of single-base exchanges in segments of DNA
(Fischer and Lerman, 1979). Gels are prepared with a
gradient from no additive to 7 mol L21 urea and 40%
formamide, and run at approximately 608C. The dierences in melting cause two fragments of DNA, which slow
down at dierent levels of the gel. The obtained pattern
displays single-base dierences. See also: Nucleic Acids:
Thermal Stability and Denaturation

Temperature gradient gel electrophoresis

Similar eects to DGGE can be achieved with temperature
gradient gel electrophoresis (Riesner et al., 1989). In this
technique, denaturing gels are run on a dierentially thermostated plate with a cold side (158C) at the cathode and a
hot side (608C) at the anode. The technique is mainly used
for screening purposes. See also: Capillary Electrophoresis;
Genomic DNA: Purication

References
Ansorge W and Labeit S (1984) Field gradients improve resolution on DNA sequencing gels. Journal of Biochemical and
Biophysical Methods 10: 237243.
Fischer SG and Lerman LS (1979) Two-dimensional electrophoretic separation of restriction enzyme fragments of DNA.
Methods in Enzymology 68: 183191.
Goldman D and Merril CR (1982) Silver staining of DNA in
polyacrylamide gels: linearity and eect of fragment size.
Electrophoresis 3: 2426.
Noolandie J, Slater DW, Lim HA and Viovy JL (1989) Generalized tube model of biased reptation for gel electrophoresis of
DNA. Science 243: 14561458.
Riesner D, Steger G, Zimmat R et al. (1989) Temperature-gradient electrophoresis of nucleic acids: analysis of conformational transitions, sequence variations, and proteinnucleic
acid interactions. Electrophoresis 10: 377389.
Sanger F and Coulson AR (1978) The use of thin acrylamide gels
for DNA sequencing. FEBS Letters 87: 107110.
Schwartz DC and Cantor CR (1984) Separation of yeast
chromosome-sized DNA by pulsed eld gradient gel electrophoresis. Cell 37: 6775.
Southern EM (1975) Detection of specic sequences among DNA
fragments separated by gel electrophoresis. Journal of
Molecular Biology 98: 503517.

Further Reading
Bova R and Micheli MR (eds) (1997) Fingerprinting Methods
Based on PCR. Heidelberg: Springer.
Landegren U (ed.) (1996) Laboratory Protocols for Mutation
Detection. Oxford, UK: Oxford University Press.
Martin R (1996) Gel Electrophoresis: Nucleic Acids. Oxford, UK:
Bios Scientic Publishers.
Rickwood D and Hames BD (eds) (1982) Gel Electrophoresis of
Nucleic Acids. Oxford, UK: IRL Press.
Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY:
Cold Spring Harbor Laboratory Press.
Westermeier R (2004) Electrophoresis in Practice, 4th edn.
Weinheim: WILEY-VCH.

eLS & 2013, John Wiley & Sons, Ltd. www.els.net