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Sep 15, 2016 - Int J Clin Exp Pathol 2016;9(9):9497-9502 www.ijcep.com /ISSN:1936-2625/IJCEP0035673. Original Article. Application of pulsed-field gel electrophoresis .....  Shueh CS, Neela V, Hussin S, Hamat RA. Sim- ple, time saving pulsed-fiel
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Two-dimensional gel electrophoresis in proteomics: past, present and future. Thierry Rabilloud. 1,2. , Mireille .... period, such as the number of proteins present in a mammalian cell  or the knowledge that serum ...... Proceedings of the Nationa
Nov 8, 2005 - A blind test on soils from a crime, an alibi scene and unrelated locations was conducted to evaluate the potential of environmental PCR and denaturating gradient gel electrophoresis for use in forensic science. In most cases, soil patte
Prepare an adequate volume of electrophoresis buffer (TAE or TBE; see Critical. Parameters, Electrophoresis buffers) to fill the electrophoresis tank and prepare the gel. To facilitate visualization of DNA fragments during the run, ethidium bromide s
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 classes of molecules can be run on each type of gel?
Which forces are influencing the molecules during their migration in the gels?
What is the concept of restriction fragment analyses?
How can large quantities of proteins of interest be obtained?
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. 1.1. Base pairs There are four heterocyclic bases which are found in deoxyribonucleic acid (DNA): two purine bases, adenine (A) and guanine (G), and two pyrimidine bases, thymine (T) and cytosine (C). Each base can pair up specifically with another base — adenine with thymine (A–T) and guanine with cytosine (G–C). This base pairing is facilitated by two or three hydrogen bonds.
Figure 1: the base pairs
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).
Figure 2: the DNA helix
1.3. Gene The instructions stored within every living cell are its genes. A gene is defined as the segment of DNA sequence corresponding to the production of a single protein (or single catalytic or structural RNA molecule). Other parts of the DNA sequence can display structural purposes or are involved in regulating 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.
1.5. Proteins Figure 3: from gene to protein Proteins are partly structural—as in connective tissue—and partly functional—as in enzymes, the catalysts for biological reactions. Each cell contains many different protein molecules which, aside from water, make up for most of its mass. The structure of proteins may be divided into four levels of organisation. Primary structure: a simple amino acid sequence linked by peptide bonds. Secondary structure: The folding of parts of the primary structure into either α helices or β sheets. Tertiary structure: The full 3D organisation of the polypeptide chain. Quaternary structure: The complete structure of a protein with more than one polypeptide chain. The destruction or rearrangement of the quaternary and tertiary, and in some cases the secondary, structure is known as denaturation.
Gel Electrophoresis, introduction A molecule with a net charge will migrate in an electric field. This phenomenon, termed electrophoresis, offers a powerful means of separating macromolecules, such as proteins, DNA and RNA. The velocity of migration (v) of a molecule in an electric field depends on the electric field strength (E) and on the electrophoretic mobility (µ) of the molecule (Equation 1). 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. Intermediatesize molecules move through the gel with various degrees of facility. In the case of gel electrophoresis, the following relation is found: Equation 2:
log µ = log µ0 − K rτ
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
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
DNA migration a
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. The Gel 3.3.1. Structure of the Gel Agarose, which is extracted from seaweed, is a linear polymer whose basic structure is based on the alternate structure of β-D-galactose units and 3,6-anhydro-α-L-galactose units (Figure 5). The linear strains form double helices, which are extensively aggregated. These aggregations provide the structural frame for the gel network. A typical gel contains 0.5-2% of agarose. 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.
Figure 5: Structure of an agarose gel a. Gel b. schematic representation of the gel network c. basic structure of the monomer
CH2OH O O O OH
c 3.4. Factors affecting the rate of DNA migration Different factors affect the velocity of DNA (v) molecules in the gel by acting on the parameters of the equation 1 and 2: 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]) 0.3 0.6 0.7 0.9 1.2 1.5 2.0
Efficient range of separation of linear DNA molecules (kb) 5-60 1-20 0.8-10 0.5-7 0.4-6 0.2-3 0.1-2
Table 1: Range of separation in gels containing different amounts of agarose (1 kb = 1 kilobase = 1000 nucleotides)
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). D. Conformation of the DNA Circular and linear DNAs of the same molecular weight migrate through agarose gels at different rates (shape 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.
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.
3.6. Example of application: Restriction fragment analysis
DNA cutted by a restriction enzyme
Each restriction enzyme recognises a short, specific sequence of nucleotide bases (called restriction site). When an endonuclease identifies a restriction site, it catalyses the hydrolysis of the sugar-phosphate bond linking adjacent nucleotides (Figure 7).
3.6.1. Restriction Enzymes Restriction enzymes are proteins produced by bacteria that cleave DNA at specific sites along the molecule. They can be seen as scissors for DNA that only cut if they detect a specific order of base pairs. In the bacterial cell, restriction enzymes cleave foreign DNA, thus eliminating infecting organisms. Restriction enzymes can be isolated from bacterial cells and used to manipulate fragments of DNA. For this reason they are indispensable tools of recombinant DNA technology. A recombinant DNA molecule is an artificial DNA molecule created in the test tube by ligating ("gluing") together pieces of DNA. Plasmids are circular DNA molecules coding for one or several genes found in bacteria. Recombinant plasmids, i.e. plasmid engineered in the test tube, are widely used in biotechnology. They allow the introduction of recombinant genes in bacteria.
5kb 3kb 2kb 1.5kb 1kb 0.75kb 0.5kb 0.3kb 0.1kb
Agarose Gel after electrophoresis. Each band is a DNA fragment
3.6.2. Restriction fragments analysis The digestion of a linear DNA fragment containing one restriction site with the corresponding enzyme will produce two restriction fragments, whereas a plasmid (circular molecule) will be linearised. The analysis of the restriction fragments allows to conclude at which site of the DNA sequence the enzymes cut. Example: determining the position of two restriction sites (A and B) of a 3.5kb DNA fragment. Digestions products are analysed on an agarose gel (Figure 7). Lane 1 is a ladder giving the reference length. Lane 2 shows the intact fragment. Lane 3 shows the products of the digestion by enzyme A. Lane 4 shows the products of digestion by enzyme B. Lane 5 shows the products of the digestion by enzyme A and B together (double digestion).
Figure 7: 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
The products of the double digestion by A and B (lane 5), indicate that the left hand map is the correct one.
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). 4.2. The Gel 4.2.1. Structure of the gel The gel is formed by the linear radical polymerisation of acrylamide crosslinked by N,N’-methylene-bisacrylamide.
O HN O
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. 4.2.3. Non denaturing gels Proteins can be separated in their native (functional) form. The electrophoretic mobility of native proteins is strongly dependant on the shape (tertiary and quaternary structures) and their net charge (dependent on the pH of the gel). These gels are mainly used as preparative gels and the intact and often functional proteins can then be extracted and used for bioassays. 4.2.4. Denaturing gels After denaturation, all proteins have a similar shape and only the primary structure (the linear sequence of amino acids) remains different. The shape contribution to Kr is then equal for all proteins and the migration is only dependent on the weight and the net charge of the protein. To achieve a PAGE in these conditions, a denaturing agent has to be added to the gel. 8
4.2.5. SDS-PAGE Proteins (in contrast to DNA) do not have a fixed mass-to-charge ratio (in DNA, there is one negative charge per nucleotide). This is why, in their native form, proteins cannot be separated by size using electrophoresis. Thus almost all analytical electrophoresis of proteins is carried out under denaturing conditions and in presence of the strong anionic detergent SDS (sodium dodecyl sulphate). Because SDS is an anionic detergent, when it binds proteins, it coats them with negative charges (Figure 9). When proteins are coated with negatively charged SDS molecules, an artificial mass-to-charge uniformity is created- the larger the polypeptide, the larger the number of SDS molecules needed to cover it- and µ0 is (almost) the same for all SDS-polypeptides. Mixtures of SDS-denatured proteins will migrate through a polyacrylamide gel with a speed based on their relative molecular weights. By using markers of known molecular weight, it is therefore possible to estimate the molecular weight of the polypeptide chains. + -
+ Native protein
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
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
Linear range of separation (kDa) 12-43 16-68 36-94 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. 4.5.1. Staining resulting from a chemical reaction In the silver staining, Ag+ ions form complexes with the Glutamate, Asparate and Cystein residues of the proteins. Alkaline formaldehyde reduces the bound Ag+ from the complexes to Ag. The details of this reaction are still unknown. The precipitated Ag reveals the protein in the gel. 4.5.2. Staining with protein binding dyes Different dyes that bind to the proteins can be used. The most common is Coomassie brilliant blue. It binds to proteins via physisorption to arginine, histidine and the aromatic amino acids (Figure 11). The commassie staining is quantitative. High molecular weight
Low molecular weight
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
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 produce this protein in very high quantities (Figure 12). Bacteria culture Bacteria culture + chemical inducing the production of the protein coded in the plasmid
Normal bacterial proteins
Normal bacterial proteins + the recombinant protein
The SDS-PAGE is then the method of choice to check the production of the protein. One can run the protein extract of induced bacteria cultures in parallel with the extract of a non-induced culture. With the help of the standard ladder, the presence of more intense bands at the corresponding molecular weight can be checked.
Figure 12: Recombinant protein induction and analysis by SDS-PAGE
Goals of this practical work During this practical work you will digest a plasmid with different enzymes and perform a restriction fragment analysis to draw the plasmid map . You will visualize the protein extracts of recombinant bacteria cultures by SDS-PAGE. You will also run protein extracts from different organisms to visualize the difference in protein pattern.
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 a) Sample Preparation Due to the long preparation time these samples have been prepared by the supervisor before the practical. 1
Restriction fragment analysis
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 gel. Then raise the voltage to 250 V. Run the gel at this voltage until the migration front reaches the end of the gel.
2 a) Sample preparation You will perform 3 single digestions and 3 double 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 Incubate the samples at 37°C for 1 hour. Some double digestions require two digestions of 30 min.
3 d) Staining Disassemble the apparatus and take out the gel. Place it in the staining solution (Coomassie blue). Warning! Contains MeOH and Acetic acid (Use a fume hood). Stain for 30 min on the shaker.
4 c) Loading Add the corresponding volume of 6x loading buffer to the digestions. Also prepare an uncut plasmid sample. Load the samples and a ladder on the agarose (1%) gel. Warning! The gel contains ethidium bromide
5 e) Destaining Transfer the gel (under the fume hood) into the destaining solution. Exchange the destaining solution every ~10min, until the protein bands are clearly visible.
d) Gel running (~40 min) Run the gel at 100 V until the migration front (blue) reaches the middle of the gel.
6 f) Scan the gel Place the gel in a plastic wrap and scan it.
e) Observe the gel on the UV table. The supervisor 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
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?
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 Der Experimentator: Proteinbiochemie/Proteomics, 4th edition, Rehm, Spektrum Akademischer Verlag, 2002 How does an SDS PAGE really work, www.mullinslab.ucsf.edu/protocols/html/SDS_PAGE_protocol.htm 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/