Gel electrophoresis
Gel electrophoresis is a widely used laboratory technique that enables the separation and analysis of biomolecules such as DNA, RNA, and proteins based on their size. This method relies on the principle that smaller molecules can move more easily through a gel matrix than larger ones when an electric field is applied. Typically, the gel is made from agarose or acrylamide, which creates a porous structure that facilitates the movement of these molecules.
In practice, samples are loaded into wells created in the gel, and an electric current is applied, causing negatively charged nucleic acids to migrate toward the positive electrode. The distance each molecule travels is proportional to its size, allowing researchers to estimate their molecular weight. Visualization of the separated molecules is achieved using specific dyes: ethidium bromide for nucleic acids and Coomassie blue for proteins, which bind to the respective molecules and enable their detection under ultraviolet light or in visible wavelengths.
Gel electrophoresis is essential for various applications in molecular biology, including genetic research, protein analysis, and diagnostics, making it a crucial tool for scientists across disciplines.
Gel electrophoresis
Significance: Gel electrophoresis is a laboratory technique involving the movement of charged molecules in a buffer solution when an electric field is applied to the solution. The technique allows scientists to separate DNA, RNA, and proteins according to their size. The method is the most widely used way to determine the molecular weight of these molecules and can be used to determine the approximate size of most DNA molecules and proteins.
Basic Theory of Electrophoresis
Biologists often need to determine the approximate size of DNA fragments, RNA, or proteins. All of these molecules are much too small to visualize using conventional methods. The size of a piece of DNA capable of carrying all the information needed for a single gene may be only 2 microns long and 20 angstroms wide, while the protein encoded by this gene might form into a globular ball only 2.5 to 10 nanometers in diameter. Therefore, some indirect method of “seeing” the length of these molecules must be used. The easiest and by far most common way to do this is by gel electrophoresis. Electrophoresis is based on the theory that if molecules can be induced to move in the same direction through a tangled web of material, smaller molecules will move farther through the matrix than larger molecules. Thus, the distance a molecule moves will be related to its size, and knowing the basic chemical nature of the molecule will allow an approximation of its relative molecular weight.
As an analogy, imagine a family with two children picnicking by a thick, brushy forest. Their small dog runs into the brush, and the whole family runs in after it. The dog, being the smallest, penetrates into the center of the forest. The six-year-old can duck through many of the branches and manages to get two-thirds of the way in; the twelve-year-old makes it halfway; the mother gets tangled up and must stop after only a short distance; the father, too large to fit in anywhere, cannot enter at all. This is what happens to molecules moving through a gel: Some travel through unimpeded, others are separated into easily visualized size groups, and others cannot even enter the matrix.
The Electrophoresis Setup
The gel is typically composed of a buffer solution containing agarose or acrylamide, two polymers that easily form a gel-like material at room temperature. At first the buffer/polymer solution is liquid and is poured into a casting chamber composed of a special tray or of two plates of glass with a narrow space between them. A piece of plastic with alternating indentations like an oversized comb is pushed into one end of the gel while it is still liquid. When the gel has solidified, the “comb” is removed, leaving small depressions in the matrix (wells) into which the DNA, RNA, or protein sample is applied. The gel is then attached to an apparatus that exposes the ends of the gel to a buffer, each chamber of which is attached to an electric power supply. The buffer allows an even application of the electric field.
Since the molecules of interest are so small, matrices with small pore size must be created. It is important to find a matrix that will properly separate the molecules being studied. The key is to find a material that creates pores large enough to let DNA or proteins enter but small enough to impede larger molecules. By using different concentrations of agarose or acrylamide, anything from very short pieces of DNA that differ only by a single nucleotide to whole chromosomes can be separated.
Agarose is composed of long, linear chains of multiple monosaccharides (sugars). At high temperatures, 95 degrees Celsius (203 degrees Fahrenheit), the agarose will “melt” in a buffer solution. As the gel cools to around 50 degrees Celsius (122 degrees Fahrenheit), the long chains begin to wrap around each other and solidify into a gel. The concentration of agarose determines the pore size, since a larger concentration will create more of a tangle. Agarose is usually used with large DNA or RNA molecules.
Acrylamide is a short molecule made up of a core of two carbons connected through a double bond with a short side-chain with a carboxyl and amino group. When the reactive chemicals ammonium persulfate and TEMED are added, the carbon ends fuse together to create long chains of polyacrylamide. If this were the only reaction, the end result would be much like agarose. However, a small number (usually 5 percent or less) of the acrylamides are the related molecule called bis-acrylamide, a two-headed version of the acrylamide molecule. This allows the formation of interconnecting branch points every twenty to fifty acrylamide residues on the chain, which creates a pattern more like a net than the tangled strands of agarose. This results in a narrower pore size than agarose, which allows the separation of much smaller fragments. Acrylamide is used to separate proteins and small DNA fragments and for sequencing gels in which DNA fragments differing in size by only a single nucleotide must be clearly separated.
Why Nucleic Acids and Proteins Move in a Gel
DNA and RNA will migrate in an electric field since every base has a net negative charge. This means that DNA molecules are negatively charged and will migrate toward the positive pole if placed in an electric field. In fact, since each base contributes the same charge, the amount of negative charge is directly proportional to the length of the DNA. This means that the electromotive force on any piece of DNA or RNA is directly proportional to its length (and therefore its mass) and that the rate of movement of DNA or RNA molecules of the same length should be the same.
The charge on different amino acids varies considerably, and the proportions of the various amino acids vary widely from protein to protein. Therefore, the charge on a protein has nothing to do with its length. To correct for this, proteins are mixed with the detergent sodium dodecyl sulfate, or SDS (the same material that gives most shampoos their suds), before being loaded onto the gel. The detergent coats the protein evenly. This has two important effects. The first is that the protein becomes denatured, and the polypeptide chain will largely exist as a long strand (rather than being compactly bunched, as it normally is). This is important because a tightly balled protein would more easily pass through the polyacrylamide matrix than a linear molecule, and proteins with the same molecular weight might appear to be different sizes. More important, each SDS molecule has a slight negative charge, so the even coating of the protein results in a negative charge that is directly proportional to the size of the protein.
Once the molecules have been subjected to the electric field long enough to separate them in the gel, they must be visualized. This is done by soaking the gel in a solution that contains a dye that stains the molecules. For DNA and RNA, this dye is usually ethidium bromide, a molecule that has an affinity for nucleic acids and slips between the strands or intercalates into the helix. The dye, when exposed to ultraviolet light, glows orange, revealing the location of the nucleic acid in the gel. For proteins, the dye Coomassie blue is usually used, a stain that readily binds to proteins of most types.
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