Chapter 4: Electrophoresis - Introduction

Figure 4.1 Hoefer SE 400 Sturdier Electrophoresis units

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. 1

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 4.1 shows a typical slab electrophoresis unit.

When the detergent SDS (sodium dodecyl sulfate) 2 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.

Polyacrylamide gels are formed from the polymerization of two compounds, acrylamide and N,N^1-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^1,N^1,- 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

Figure 4.2 Electrophoretic separations of proteins

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 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 4.2 presents a typical protein separation pattern.

Continuous vs discontinuous gel systems

Figure 4.3 Schematic diagram of electrophoresis

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, 4 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 bands. The bands then are separated from each other on a discontinuous (i.e. disc ) pH gel. 5

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 (Tris-Glycine). The leading ion is usually Cl¯ glycinate is the trailing ion. A schematic of this anionic system is shown in Figure 4.3. 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

Figure 4.4 Agarose separation of cDNA

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 10^6 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 4.4 demonstrates the separation of DNA fragments on an agarose gel.

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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 --