Professional Documents
Culture Documents
Gel Electrophoresis
Separating DNA and Proteins
Supervisors
Charles Vidoudez, charles.vidoudez@uni-jena.de, 03641-948178
Matthew Welling, matthew.welling@uni-jena.de, 03641-948178
IAAC, Lehrstuhl für instrumentelle Analytik, Lessingstr. 8, Raum 306, 07743 Jena
At the beginning of the practical the theoretical introduction will be discussed. Please consider the
following control questions:
• What are the major differences between agarose gels and polyacrylamide gels? What are their
advantages and disadvantages?
• Which forces are influencing the molecules during their migration in the gels?
1
1. From the genetic code to proteins
All living cells on Earth store their information in the form of double-stranded molecules of DNA, which
provide the blueprints for proteins. In order to understand this code of life analytical methods were
developed that allow separation of these biomacromolecules. This lab class addresses the fundamental
methods currently employed for DNA and protein separation.
1.2. DNA
When bound to a sugar and a phosphate, the bases form a nucleotide. A single strand of DNA is a
polymer consisting of nucleotide monomers joined together by sugar-phosphate linkages. Each polymeric
strand of DNA coils up into a helix and is bonded to another complimentary strand by hydrogen bonds
between the bases. The resulting double stranded helix of DNA can adopt either a linear or circular shape
and is usually described in terms of the number of base pairs (bp, 1000bp = kb).
2
the use of the genetic information. However, there are still sequences whose purposes have not yet been
discovered.
1.4. RNA
During transcription, the sequence of the gene is copied into a ribonucleic
acid (RNA) nucleotide sequence. The RNA is complimentary to the DNA
sequence but only exists as a single strand. This then instructs the cell
machinery on protein synthesis (translation) using three nucleotide codes
to indicate different amino acids.
Equation 1: v = µE
The electrophoretic mobility is a parameter unique for each molecule and each medium. Electrophoretic
separation is nearly always carried out in gels due to their ability to serve as a molecular sieve that
enhances separation by modifying µ. Molecules that are small compared to the pores in the gel readily
move through the gel, whereas molecules much larger than the pores are almost immobile. Intermediate-
size molecules move through the gel with various degrees of facility.
In the case of gel electrophoresis, the following relation is found:
where µ0 is the free electrophoretic mobility of the molecule (mobility in a non-sieving medium), Kr the
retardation coefficient and τ the concentration of the gel. µ0 is dependant on the mass-to-charge ratio of
the molecule, whereas Kr is related to the propriety of the gel, the size and the shape of the migrating
molecule.
The matrix used for gel electrophoresis is either a cross-linked polymer such as polyacrylamide, or a
linear polymer such as agarose.
3
3. Agarose Gel Electrophoresis
3.1. Application
Agarose gels are the standard method used to separate, identify and purify DNA (and RNA). Their
resolving power is lower than polyacrylamide gels but the range of separation is greater and they are
easier to prepare. DNA fragments ranging from 200bp up to 50kb can be separated. They can also be used
as a tool to isolate a defined DNA fragment or as a preparative step in southern blot (DNA specific
detection) or northern blot (RNA specific detection).
3.2. Apparatus
The most commonly used configuration is a horizontal slab gel. The gel is poured on a glass or plastic
tray that can be installed on a platform in the electrophoresis tank. Electrophoresis is carried out with the
gel submerged just beneath the surface of a buffer. The resistance of the gel to electric current is almost
the same as that of the buffer, and so a considerable fraction of the applied current passes along the length
of the gel (Figure 4).
Buffer Well Agarose Gel
- +
-
DNA migration
DNA migration
Platinum Electrodes +
a b
Figure 4: Agarose Gel Electrophoresis apparatus a. sideview b. top down view.
The row of wells allows samples to be run in parallel, including a standard ladder.
Samples are inserted into wells placed at one end of the gel. In the buffer (pH = 7.5-7.8) DNA is
negatively charged due to deprotonation of the phosphate linkage and will migrate towards the cathode.
3.3.2. Preparation
Agarose powder is melted in a buffered solution by boiling. The solution is poured into a gel tray and
polymerisation occurs as it cools to room temperature.
4
b
a
A. Voltage applied
The voltage determines directly the electric field strength (E) of the Equation 1. But only at low
voltages the rate of migration of linear DNA fragments is proportional to the voltage applied. At
high voltages, µ is also significantly affected and modifies the migration speed. To obtain
maximum resolution of DNA fragments, the gel should be run at no more than 5V/cm (distance
between the electrodes).
B. Agarose concentration
According to Equation 2, there is a linear relationship between the logarithm of the electrophoretic
mobility of DNA and the agarose concentration in the gel. Thus, by using gels of different
concentrations, it is possible to resolve a wide range of DNA molecules (Table 1).
Amount of agarose in gel (%[w/v]) Efficient range of separation of linear DNA
molecules (kb)
0.3 5-60
0.6 1-20
0.7 0.8-10
0.9 0.5-7
1.2 0.4-6
1.5 0.2-3
2.0 0.1-2
Table 1: Range of separation in gels containing different amounts of agarose (1 kb = 1 kilobase = 1000 nucleotides)
5
C. Molecular size of the DNA
The electrophoretic mobility of linear double-stranded DNA is inversely proportional to the log10
of the number of base pairs (size component of Kr).
E. Electrophoresis Buffer
The electrophoretic mobility of DNA is affected by the composition and ionic strength of the
electrophoresis buffer. The mass-to-charge ratio, constant for all DNA molecules in a given
condition, is dependant on the pH of the buffer.
The buffering capacity is also important, as the buffer tends to become exhausted during extended
electrophoresis.
The usual buffers contain EDTA and Tris-acetate (TAE), Tris-borate (TBE) or Tris-phosphate
(TPE) at a concentration of approximately 50 mM (pH 7.5-7.8). DNA degrading enzymes
(nucleases) are inhibited by the addition of EDTA. This traps Mg2+ ions that are required by these
enzymes.
F. Intercalating dyes
Ethidium bromide (see 3.5), by affecting the shape of DNA and therefore Kr, reduces the
electrophoretic mobility of linear DNA by about 15%.
3.5. Staining
The most convenient method to visualize DNA in agarose gels is staining with the fluorescent dye
ethidium bromide.
Br- N+
H2N NH2
The planar group intercalates between the stacked bases of DNA. Ultraviolet
radiation at 302 nm and 366 nm is absorbed by the bound dye and the energy
is re-emitted at 590 nm in the red-orange region of the visible spectrum.
Because the fluorescence of the bound dye is about 20 times greater than that
of the unbound one, small amounts of DNA can be detected in the presence of
free ethidium bromide in the gel (Figure 6). The detection limit with this
staining is about 2ng of DNA. The main disadvantage of ethidium bromide is
its toxicity and powerful mutagenicity.
Figure 6 Ethidium Bromide structure (up left) and intercalated in DNA (up right). Picture: UV revealing of the ethidium
bromide bound DNA fragments in an agarose gel.
6
3.6. Example of application: Restriction fragment analysis
By comparing the length of the fragments on lanes 3 and 4, the two following positions of restriction
sites A and B are possible:
2kb 1kb 0.5kb 1.5kb 1.5kb 0.5kb
B A B A
The products of the double digestion by A and B (lane 5), indicate that the left hand map is the correct
one.
7
4. PolyAcrylamide Gel Electrophoresis (PAGE)
4.1. Application
Proteins can be separated efficiently on polyacrylamide gels. PAGE of proteins is used as a preparative
method to separate proteins before further analysis but it can also be used as a direct analytical tool by
revealing the mass of the separated proteins directly in the gel (see 4.5.2).
O NH2 O NH2
O
NH2 O N N O
H H
O
a Acrylamide N,N’-methylene-bis-acrylamide O NH2 O NH2
HN
O NH2 HN O
NH2
O
b c O NH2 O NH2
Figure 8: Polyacrylamide gel. a. Structure of acrylamide and bisacrylamide. b. Gel pressed between the two glass holding
plates. c. Details of the crosslinked polymer.
The concentration of acrylamide in buffer ranges from 5% to 20% and is used to determine the size of the
pores. The ratio between acrylamide and bis-acrylamide ranges from 20:1 to 30:1, the most common ratio
being 29:1.
4.2.2. Preparation
A mixture of acrylamide and bisacrylamide is prepared in a buffer. Ammonium persulfate is added to
provide the free radicals initiating the polymerisation. The solution is then directly poured between two
glass plates to give the shape of the gel and to hold it in place.
Figure 9: SDS and its effect. Left, SDS structure. Right, action of SDS on a protein (blue line).
4.3. Apparatus
In contrast to the agarose gel electrophoresis, PAGE is usually run vertically (Figure 10). SDS-PAGE is
carried out with two different types of polyacrylamide gels fused together. The gel is thus composed of
two parts, the "stacking gel" and the "resolving gel". The role of the stacking gel is to concentrate the
protein mix loaded in the wells into a sharp band before it enters the resolving gel.
Loading well
Glas plates
Anode
Cathode
9
4.4. Factor influencing migration
The major factors influencing the migration of the SDS-Protein complexes in the resolving gel, apart
from the molecular weight, are the acrylamide-bisacrylamide ratio (affecting the “gel proprieties”
component of Kr) and the total polymer concentration (τ).
Cross-links formed from bisacrylamide add rigidity and tensile strength to the gel and form pores through
which the SDS-polypeptide complexes must pass. The size of these pores decreases as the
bisacrylamide:acrylamide ratio increases, reaching a minimum when the ratio is approximately 1:20.
Most SDS-Polyacrylamide gels are cast with a molar ratio of 1:29, which has been shown empirically to
be capable of resolving polypeptides that differ in size by as little as 3%.
Table 2 shows the linear range of separation obtained with gels containing 5% to 15% of polymer.
Table 2: Effective range of separation of SDS-Polyacrylamide gels, with a bisacrylamide:acrylamide ratio of 1:29
Acrylamide polymer concentration (%) Linear range of separation (kDa)
15 12-43
10 16-68
7.5 36-94
5.0 57-212
4.5. Staining
To reveal the protein in the polyacrylamide gels, different staining techniques have been developed. Two
different approaches can be used.
Figure 11: Left SDS-polyacrylamide gel stained with Coomassie blue. The first lane is a standard ladder. The other lanes
present protein mixes separated. Right: structure of Coomassie Blue
10
4.6. Example of SDS-PAGE application – Check of the induced production of a recombinant protein
When one wants to study a protein, recombinant DNA
technology can be used. A plasmid can be engineered to
contain the gene coding for the protein of interest. A
bacterium containing this plasmid can be induced to
Bacteria culture Bacteria culture produce this protein in very high quantities (Figure 12).
+
chemical inducing the production The SDS-PAGE is then the method of choice to check
of the protein coded in the plasmid the production of the protein. One can run the protein
extract of induced bacteria cultures in parallel with the
Normal bacterial Normal bacterial proteins
extract of a non-induced culture. With the help of the
proteins + the recombinant protein standard ladder, the presence of more intense bands at
the corresponding molecular weight can be checked.
SDS-PAGE
11
6. Protocols
The restriction analysis and the SDS-PAGE need long gel running time, so both experiments will be
conducted in parallel. Each of the two following columns describes one of the experiments. The framed
numbers indicate in which order the steps will be conducted during the practical.
SDS-PAGE Restriction fragment analysis
a) Sample Preparation
Due to the long preparation time these samples
have been prepared by the supervisor before the
practical.
1
b) Sample Loading
Load the 5 samples (30µL) on the polyacrylamide
gel (5%/10%) with the Eppendorf pipette. Note the
order of loading.
c) Gel running (~1hour)
Run the gel at 100 V until the migration front
(blue) reaches the interface stacking-resolving 2
gel. Then raise the voltage to 250 V.
a) Sample preparation
Run the gel at this voltage until the migration front
You will perform 3 single digestions and 3 double
reaches the end of the gel.
digestions.
Calculate the volume of buffer, plasmid solution,
water and enzyme to mix, according to the
concentration given by the supervisor during the
practical.
Prepare the digestions
b) Digestion
3 Incubate the samples at 37°C for 1 hour. Some
d) Staining double digestions require two digestions of 30 min.
Disassemble the apparatus and take out the gel.
Place it in the staining solution (Coomassie blue).
4
Warning! Contains MeOH and Acetic acid (Use a
fume hood). c) Loading
Stain for 30 min on the shaker. Add the corresponding volume of 6x loading buffer to
5 the digestions. Also prepare an uncut plasmid sample.
Load the samples and a ladder on the agarose (1%)
e) Destaining
gel. Warning! The gel contains ethidium bromide
Transfer the gel (under the fume hood) into the
destaining solution. Exchange the destaining d) Gel running (~40 min)
solution every ~10min, until the protein bands are Run the gel at 100 V until the migration front (blue)
clearly visible. reaches the middle of the gel.
6
f) Scan the gel e) Observe the gel on the UV table. The supervisor
Place the gel in a plastic wrap and scan it. will take a picture. The bands lower than 500 bp
are difficult to see or even not detectable.
Notice the migration of the ethidium bromide in
the gel.
12
7. Analysis and report
The report can be written in English (recommended) or in German.
Write a short introduction summarising the important concepts and use of gel electrophoresis (no
equations and not copied from the theory part).
Describe shortly what you did in the practical, including samples loading order, digestions you made
(which enzymes, buffers, concentration).
Make a table with the restriction fragment lengths you estimated from the gel, for each digestion.
According to this table, draw the map of the plasmid for the corresponding restriction sites.
Estimate the molecular weight of the recombinant protein and analyse the different protein patterns.
And in your descriptions of the practical part or the analysis, answer the following questions:
The loading buffers contain a dye and glycerol. Why?
How does ethidium bromide migrate in the agarose gel? Why?
Why is the digestion temperature 37°C?
8. References
Molecular Cloning, A laboratory Manual, 2nd edition, Sambrook, Fritsch & Maniatis, Cold Spring Harbor
Laboratory Press, 1989.
The agarose double helix and its function in agarose gel structure, Arnott et al., Journal of Molecular
Biology, 1974, 90, p. 269
Molecular Biology of the Cell, 4th edition, Alberts, Garland Science, 2002
Gene cloning & DNA analysis, 5th edition, Brown, Blackwell Publishing, 2006
Organic Chemistry, Jonathon Clayden et al., 1st Ed., Oxford University Press, 2001.
http://www.bio.miami.edu/
http://www.ornl.gov/
13