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Electrophoresis

Introduction Electrophoresis may be the main technique for molecular separation in today's cell biology laboratory. Because it is such a powerful technique, and yet reasonably easy and inexpensive, it has become commonplace. In spite of the many physical arrangments for the apparatus, and regardless of the medium through which molecules are allowed to migrate, all electrophoretic separations depend upon the charge distribution of the molecules being separated. Electrophoresis can be one dimensional (i.e. one plane of separation) or two dimensional. One dimensional electrophoresis is used for most routine protein and nucleic acid separations. Two dimensional separation of proteins is used for finger printing , and when properly constructed can be extremely accurate in resolving all of the proteins present within a cell (greater than 1,500). The support medium for electrophoresis can be formed into a gel within a tube or it can be layered into flat sheets. The tubes are used for easy one dimensional separations (nearly anyone can make their own apparatus from inexpensive materials found in any lab), while the sheets have a larger surface area and are better for two- dimensional separations. Figure 1 shows a typical slab electrophoresis unit. When the detergent SDS (sodium dodecyl sulfate) is used with proteins, all of the proteins become negatively charged by their attachment to the SDS anions. When separated on a polyacrylamide gel, the procedure is abbreviated as SDS--PAGE (for Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis). The technique has become a standard means for molecular weight determination.

Figure 1 Hoefer SE 400 Sturdier Electrophoresis units

Polyacrylamide gels are formed from the polymerization of two compounds, acrylamide and N,N -methylene- bis-acrylamide (Bis, for short). Bis is a cross-linking agent for the gels. The polymerization is initiated by the addition of ammonium persulfate along with either dimethyl amino-propionitrile (DMAP) or N,N,N ,N ,- tetramethylethylenediamine (TEMED). The gels are neutral, hydrophillic, three-dimensional networks of long hydrocarbons crosslinked by methylene groups. The separation of molecules within a gel is determined by the relative size of the pores formed within the gel. The pore size of a gel is determined by two factors, the total amount of acrylamide present (designated as %T) and the amount of cross-linker (%C). As the total amount

of acrylamide increases, the pore size decreases. With cross- linking, 5%C gives the smallest pore size. Any increase or decrease in %C increases the pore size. Gels are designated as percent solutions and will have two necessary parameters. The total acrylamide is given as a % (w/v) of the acrylamide plus the bis-acrylamide. Thus, a 7 1/2 %T would indicate that there is a total of 7.5 gms of acrylamide and bis per 100 ml of gel. A gel designated as 7.5%T:5%C would have a total of 7.5% (w/v) acrylamide + bis, and the bis would be 5% of the total (with pure acrylamide composing the remaining 2.5%). Proteins with molecular weights ranging from 10,000 to 1,000,000 may be separated with 7 1/2% acrylamide gels, while proteins with higher molecular weights require lower acrylamide gel concentrations. Conversely, gels up to 30% have been used to separate small polypeptides. The higher the gel concentration, the smaller the pore size of the gel and the better it will be able to separate smaller molecules. The percent gel to use depends on the molecular weight of the protein to be separated. Use 5% gels for proteins ranging from 60,000 to 200,000 daltons, 10% gels for a range of 16,000 to 70,000 daltons and 15% gels for a range of 12,000 to 45,000 daltons. 3 Cationic vs anionic systems In electrophoresis, proteins are separated on the basis of charge, and the charge of a protein can be either + or -- , depending upon the pH of the buffer. In normal operation, a column of gel is partitioned into three sections, known as the Separating or Running Gel, the Stacking Gel and the Sample Gel. The sample gel may be eliminated and the sample introduced via a dense non-convective medium such as sucrose. Electrodes are attached to the ends of the column and an electric current passed through the partitioned gels. If the electrodes are arranged in such a way that the upper bath is -- (cathode), while the lower bath is + (anode), and -- anions are allowed to flow toward the anode, the system is known as an anionic system. Flow in the opposite direction, with + cations flowing to the cathode is a cationic system. Tube vs Slab Systems Two basic approaches have been used in the design of electrophoresis protocols. One, column electrophoresis, uses tubular gels formed in glass tubes, while the other, slab gel electrophoresis, uses flat gels formed between two plates of glass. Tube gels have an advantage in that the movement of molecules through the gels is less prone to lateral movement and thus there is a slightly improved resolution of the bands, particularly for proteins. It is also more economical, since it is relatively easy to construct homemade systems from materials on hand. However, slab gels have the advantage of allowing for two dimensional analysis, and of running multiple samples simultaneously in the same gel. Slab gels are designed with multiple lanes set up such that samples run in parallel. The size and number of the lanes can be varied and, since the samples run in the same medium, there is less likelihood of sample variation due to minor changes in the gel structure. Slab gels are

Figure 2 Electrophoretic separations of proteins

unquestionably the the technique of choice for any blot analyses and for autoradiographic analysis. Consequently, for laboratories performing routine nucleic acid analyses, and those employing antigenic controls, slab gels have become standard. The availability of reasonably priced commercial slab gel units has increased the use of slab gel systems, and the use of tube gels is becoming rare. The theory and operation of slab gel electrophoresis is identical to tube gel electrophoresis. Which system is used depends more on the experience of the investigator than on any other factor, and the availability of equipment. Figure 2 presents a typical protein separation pattern. Continuous vs discontinuous gel systems The original use of gels as separating media involved using a single gel with a uniform pH throughout. Molecules were separated on the basis of their mobility through a single gel matrix. This system has only occasional use in today's laboratory. It has been replaced with discontinous, multiple gel systems. In multiple gel systems, a separating gel is augmented with a stacking gel and an optional sample gel. These gels can have different concentrations of the same support media, or may be completely different agents. The key difference is how the molecules separate when they enter the separating gel. The proteins in the sample gel will concentrate into a small zone in the stacking gel before entering the separating gel. The zone within the stacking gel can range in thickness from a few microns to a full millimeter. As the proteins are stacked in concentrated bands, they continue to migrate into the separating gel in concentrated narrow

Figure 3Schematic diagram of electrophoresis

bands. The bands then are separated from each other on a discontinuous (i.e. disc ) pH gel. Once the protein bands enter the separating gel, separation of the bands is enhanced by ions passing through the gel column in pairs. Each ioin in the pair has the same charge polarity as the protein (usually negative), but differ in charge magnitude. One ion will have a much

greater charge magnitude than the proteins, while the other has a lesser charge magnitude than the proteins. The ion having a greater charge will move faster and is thus the leading ion, while the ion with the lesser charge will be the trailing ion. When an anionic system is employed, the Cl and glycinate (glycine as its acid derivative) ions are derived from the reservoir buffer (TrisGlycine). The leading ion is usually Cl glycinate is the trailing ion. A schematic of this anionic system is shown in Figure 3 Chloride ions enter the separating gel first and rapidly move down the gel, followed by the proteins and then the glycinate ions. The glycinate ions overtake the proteins and ultimately establish a uniform linear voltage gradient within the gel. The proteins then sort themselves within this gradient according to their charge and size. Agarose Gels While acrylamide gels have become the standard for protein analysis, they are less suitable for extremely high molecular weight nucleic acids (above 200,000 daltons). In order to properly separate these large molecules, the acrylamide concentration needs to be reduced to a level where it remains liquid. The gels can be formed, however, by the addition of agarose, a naturally linear polysaccharide, to the low concentration of acrylamide. With the addition of agarose, acrylamide concentrations of 0.5% can be used and molecular weights of up to 3.5 x 10 daltons can be separated. This is particularly useful for the separation of large sequences of DNA. Consequently, agarose-acrylamide gels are used extensively in today's genetic laboratories for the determination of gene maps. This chapter will concentrate on the separation of proteins, but Figure 4 demonstrates the separation of DNA fragments on an agarose gel.
Figure 4 Agarose separation of cDNA

Principles of Gel Electrophoresis Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge or conformation. As such, it is one of the most widely-used techniques in biochemistry and molecular biology. When charged molecules are placed in an electric field, they migrate toward either the positive or negative pole according to their charge. In contrast to proteins, which can have either a net positive or net

negative charge, nucleic acids have a consistent negative charge imparted by their phosphate backbone, and migrate toward the anode. Proteins and nucleic acids are electrophoresed within a matrix or "gel". Most commonly, the gel is cast in the shape of a thin slab, with wells for loading the sample. The gel is immersed within an electrophoresis buffer that provides ions to carry a current and some type of buffer to maintain the pH at a relatively constant value. The gel itself is composed of either agarose or polyacrylamide, each of which have attributes suitable to particular tasks: Agarose is a polysaccharide extracted from seaweed. It is typically used at concentrations of 0.5 to 2%. The higher the agarose concentration the "stiffer" the gel. Agarose gels are extremely easy to prepare: you simply mix agarose powder with buffer solution, melt it by heating, and pour the gel. It is also non-toxic. Agarose gels have a large range of separation, but relatively low resolving power. By varying the concentration of agarose, fragments of DNA from about 200 to 50,000 bp can be separated using standard electrophoretic techniques. Polyacrylamide is a cross-linked polymer of acrylamide. The length of the polymer chains is dictated by the concentration of acrylamide used, which is typically between 3.5 and 20%. Polyacrylamide gels are significantly more annoying to prepare than agarose gels. Because oxygen inhibits the polymerization process, they must be poured between glass plates (or cylinders). Acrylamide is a potent neurotoxin and should be handled with care! Wear disposable gloves when handling solutions of acrylamide, and a mask when weighing out powder. Polyacrylamide is considered to be non-toxic, but polyacrylamide gels should also be handled with gloves due to the possible presence of free acrylamide. Polyacrylamide gels have a rather small range of separation, but very high resolving power. In the case of DNA, polyacrylamide is used for separating fragments of less than about 500 bp. However, under appropriate conditions, fragments of DNA differing is length by a single base pair are easily resolved. In contrast to agarose, polyacrylamide gels are used extensively for separating and characterizing mixtures of proteins.

Agarose Gel Electrophoresis of DNA Preparing and Running Standard Agarose DNA Gels The equipment and supplies necessary for conducting agarose gel electrophoresis are relatively simple and include:

An electrophoresis chamber and power supply Gel casting trays, which are available in a variety of sizes and composed of UV-transparent plastic. The open ends of the trays are closed with tape while the gel is being cast, then removed prior to electrophoresis. Sample combs, around which molten agarose is poured to form sample wells in the gel. Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borateEDTA (TBE). Loading buffer, which contains something dense (e.g. glycerol) to allow the sample to "fall" into the sample wells, and one or two tracking dyes, which migrate in the gel and allow visual monitoring or how far the electrophoresis has proceeded. Ethidium bromide, a fluorescent dye used for staining nucleic acids. NOTE: Ethidium bromide is a known mutagen and should be handled as a hazardous chemical - wear gloves while handling. Transilluminator (an ultraviolet lightbox), which is used to visualize ethidium bromide-stained DNA in gels. NOTE: always wear protective eyewear when observing DNA on a transilluminator to prevent damage to the eyes from UV light. To pour a gel, agarose powder is mixed with electrophoresis buffer to the desired concentration, then heated in a microwave oven until completely melted. Most commonly, ethidium bromide is added to the gel (final concentration 0.5 ug/ml) at this point to facilitate visualization of DNA after electrophoresis. After cooling the solution to about 60C, it is poured into a casting tray containing a sample comb and allowed to solidify at room temperature or, if you are in a big hurry, in a refrigerator. After the gel has solidified, the comb is removed, using care not to rip the bottom of the wells. The gel, still in its plastic tray, is inserted horizontally into the electrophoresis chamber and just covered with buffer. Samples containing DNA mixed with loading buffer are then pipeted into the sample wells, the lid and power leads are placed on the apparatus, and a current is applied. You can confirm that current is flowing by observing bubbles coming off the electrodes. DNA will migrate towards the positive electrode, which is usually colored red.

The distance DNA has migrated in the gel can be judged by visually monitoring migration of the tracking dyes. Bromophenol blue and xylene cyanol dyes migrate through agarose gels at roughly the same rate as double-stranded DNA fragments of 300 and 4000 bp, respectively.

When adequate migration has occured, DNA fragments are visualized by staining with ethidium bromide. This fluorescent dye intercalates between bases of DNA and RNA. It is often incorporated into the gel so that staining occurs during electrophoresis, but the gel can also be stained after electrophoresis by soaking in a dilute solution of ethidium bromide. To visualize DNA or RNA, the gel is placed on a ultraviolet transilluminator. Be aware that DNA will diffuse within the gel over time, and examination or photography should take place shortly after cessation of electrophoresis. Migration of DNA Fragments in Agarose Fragments of linear DNA migrate through agarose gels with a mobility that is inversely proportional to the log10 of their molecular weight. In other words, if you plot the distance from the well that DNA fragments have migrated against the log10 of either their molecular weights or number of base pairs, a roughly straight line will appear Circular forms of DNA migrate in agarose distinctly differently from linear DNAs of the same mass. Typically, uncut plasmids will appear to migrate more rapidly than the same plasmid when linearized. Additionally, most preparations of uncut plasmid contain at least two topologically-different forms of DNA, corresponding to supercoiled forms and nicked circles. The image to the right shows an ethidium-stained gel with uncut plasmid in the left lane and the same plasmid linearized at a single site in the right lane. Several additional factors have important effects on the mobility of DNA fragments in agarose gels, and can be used to your advantage in optimizing separation of DNA fragments. Chief among these factors are: Agarose Concentration: By using gels with different concentrations of agarose, one can resolve different sizes of DNA fragments. Higher concentrations of agarose facilite separation of small DNAs, while low agarose concentrations allow resolution of larger DNAs. The image to the right shows migration of a set of DNA fragments in three concentrations of agarose, all of which were in the same gel tray and electrophoresed at the same voltage and for identical times. Notice how the larger fragments are much better resolved in the 0.7% gel, while the small fragments separated best in 1.5% agarose. The 1000 bp fragment is indicated in each lane. Voltage: As the voltage applied to a gel is increased, larger fragments migrate proportionally faster that small fragments. For that reason, the best resolution of fragments larger than about 2 kb is attained by applying no more than 5 volts per cm to the gel (the cm value is the distance between the two electrodes, not the length of the gel).

Electrophoresis Buffer: Several different buffers have been recommended for electrophoresis of DNA. The most commonly used for duplex DNA are TAE (Tris-acetateEDTA) and TBE (Tris-borate-EDTA). DNA fragments will migrate at somewhat different rates in these two buffers due to differences in ionic strength. Buffers not only establish a pH, but provide ions to support conductivity. If you mistakenly use water instead of buffer, there will be essentially no migration of DNA in the gel! Conversely, if you use concentrated buffer (e.g. a 10X stock solution), enough heat may be generated in the gel to melt it. Effects of Ethidium Bromide: Ethidium bromide is a fluorescent dye that intercalates between bases of nucleic acids and allows very convenient detection of DNA fragments in gels, as shown by all the images on this page. As described above, it can be incorporated into agarose gels, or added to samples of DNA before loading to enable visualization of the fragments within the gel. As might be expected, binding of ethidium bromide to DNA alters its mass and rigidity, and therefore its mobility. Isolation of DNA from Agarose and Polyacrylamide Gels In addition to its importance as an analytical tool, gel electrophoresis is widely used for isolating and then purifying specific fragments of DNA, usually in preparation for subcloning. Hence, this is a very commonly required procedure. Agarose Gels Several techniques can be used to purify DNA from agarose gels, and choosing between them is, to some extent, a matter of personal preference. They all start out by excising the desired "band" from an ethidium-stained gel viewed with a UV transilluminator. Because UV light can fragment DNA, it is best to work expeditiously and keep exposure time to a minumum. Cut out the desired piece of agarose using a razor blade or scalpel blade, and try to get as little extra agarose as possible. The block of agarose containing DNA is then subjected to any of the following. Electroelution: The block of agarose is placed in a piece of dialysis tubing with a small amount of fresh electrophoresis buffer, the ends sealed with clamps, and the bag placed into an electrophoresis chamber. Application of current will cause the DNA to migrate out of the agarose, but it will be trapped within the bag. Progress can be monitored using a transilluminator, as shown below. When the DNA is out of the agarose, the flow of current is reversed for a few seconds to knock the DNA off of the side of the tubing. The buffer containing the DNA is then collected and the DNA precipitated with ethanol.

Electroelution is more time consuming than some of the other techniques, but works well and is probably the best technique for recovery of large (> 5 kb) fragments of DNA. Binding and elution from glass or silica particles: In an environment of high salt and neutral or low pH, DNA binds avidly to glass, silica gel or diatomaceous earth. This phenomenon can be exploited to purify DNA from solutions containing impurities such as agarose. Typically, a slice of agarose containing the DNA of interest is "melted" by incubation in a solution containing chaotropic salt (e.g. sodium iodide) at a pH of 7.5 or less. Glass powder or silica gel is then added and the suspension is mixed to allow adsorption of DNA. The particles can then be recovered from the original liquid and washed by centrifugation and resuspension in high-salt-ethanol buffer. Finally, the pellet is resuspended in a solution with low or no salt at basic pH, the free particles pelleted by another centrifugation, and the DNA-containing supernate recovered. The glass or silica particles used for this technique can be prepared in house or, more conveniently, purchased from a number of suppliers. Electrophoresis onto DEAE-cellulose membranes: At low concentrations of salt, DNA binds avidly to DEAE-cellulose membranes. Fragments of DNA are electrophoresed in a standard agarose gel until they resolve adequately. One then makes a slit in the gel slightly ahead of the fragment(s) of interest and resumes electrophoresis until all of that fragment has migrated and stuck onto the membrane. The membrane is then removed, washed free of agarose in low salt buffer (150 mM NaCl, 50 mM Tris, 10 mM EDTA), then incubated for about 30 minutes at 65 C in high salt buffer (1 M NaCl, 50 mM Tris, 10 mM EDTA) to elute the DNA. Progress in binding DNA to the membrane and eluting it can be monitored with UV light to detect the ethidium bromide bound to DNA. After elution, DNA is precipitated with ethanol. This procedure is simple and provides very clean DNA. However, fragments larger than about 5 kb do not elute well from the membrane. Low melting point agarose: Agarose can be purchased that melts at about 65 C, which is well below the melting temperature of all but very small fragments of double-stranded DNA. After electrophoresing in such low melting temperature agarose, the appropriate fragment is excised, and the agarose block is added to a small quantity of buffer and incubated at 65 C to melt the agarose. An alternative technique is to digest the agarose with agarase. One can then extract the melted or digested agarose from the mixture with phenol and precipitate the DNA with ethanol.

Polyacrylamide Gels A commonly-used means of recovering DNA from polyacrylamide gels is by the socalled "crush and soak" method. The slice of polyacrylamide containing DNA is crushed in a microcentrifuge using a plastic pipet tip, and incubated with constant shaking in elution buffer (high salt) at 37C for several hours. The polyacrylamide pieces are then eliminated by centrifugation or by passing the mixture through a plug of siliconized glass wool. Finally, DNA is recovered by ethanol precipitation. DNA can also be recovered from polyacrylamide by use of certain types of silica gel particles, as described above for recovery from agarose. However, small (< 100 bp) fragments of DNA are very difficult to elute from standard glass particles.

Reference
Electrophoresis Harold Alexander Abramson New York Academy of Sciences. Section of Physical Sciences 1995