Basic Procedure, Separation Of Proteins, Separation Of Dna And Rna
Gel electrophoresis is a widely used technique for separating electrically charged molecules. It is a central technique in molecular biology and genetics laboratories, because it lets researchers separate and purify the nucleic acids DNA and RNA and proteins, so they can be studied individually. Gel electrophoresis is often followed by staining or blotting procedures used to identify the separated molecules.
Protein activity, such as for enzymes, can be determined once they are separated in the gel under conditions that do not denature the enzyme. Researchers can determine a purified protein's molecular weight by measuring how quickly the protein moves through a gel. The protein is first purified and denatured with heat and a reducing agent that disrupts disulfide bonding. It is then treated with an anionic detergent, sodium dodecyl sulfate (SDS), which disrupts the secondary, tertiary, and quaternary structure of the protein and coats it uniformly with negative charges. When run through a gel, the protein's migration rate is indirectly proportional to the logarithm of its molecular weight, so the smaller protein runs the fastest. The uniform negative charges ensure that the protein's migration rate is a function only of its molecular weight, not of whatever charges happen to be on it. Better resolution and separation can be obtained in an SDS-PAGE gel by first tightening the protein band before separating the proteins by size. This is accomplished by having the separating, or resolving, gel on the bottom and the larger pore gel, known as the stacking gel, on top. The proteins enter the top gel, where they are maintained by in a tight zone between ions generated by the electric field. This is accomplished by having ions that run both slower and faster than the negatively charged proteins, so that ions sandwich the proteins between them, tightening the protein band. The proteins leave the stacking gel and enter the separating gel. In this gel the ions no longer sandwich the proteins because of a change in pH, and the pore size is smaller so that the proteins separate by size.
If the protein is run on a gel along with a ladder of proteins of known weight, then the molecular weight of the protein can be determined by comparing its migration rate to that of proteins whose molecular weights are known. This technique is known as "sodium dodecyl sulfate-polyacrylamide gel electrophoresis," or SDS-PAGE.
Researchers can use gels to determine a protein's "isoelectric point," or the pH at which the protein's net charge is zero. Because pH changes the ionization state of several amino acid groups, the net charge on a protein is pH-dependent. By running proteins through a gel that has a pH gradient from one end to the other, this charge is gradually changed. At a certain pH, each protein's net charge will become zero, and the protein will stop moving. This procedure is known as "isoelectric focusing."
In "two-dimensional electrophoresis," a mixture of proteins is first separated in an isoelectric focusing tube gel. This tube is then placed sideways on an SDS-PAGE gel. In this way, proteins are separated based on two parameters: size and isoelectric point. Compared to techniques based on only one parameter, two-dimensional electrophoresis separates more proteins at once.
Two-dimensional electrophoresis is an important tool in proteomics. It can be used to separate large numbers of proteins that are isolated all at once after being expressed in response to a hormone, drug, or other stimulus. It can be combined with the use of DNA microarrays to allow a researcher to determine both what genes are expressed in response to a stimulus and what proteins are produced by these genes, which thereby determine an organism's physiological response to stimuli.
The techniques discussed so far are good for separating proteins and small nucleic acid fragments from 5 to 500 base pairs. The small pores of the polyacrylamide gels, however, are not appropriate for larger DNA fragments or intact DNA molecules such as plasmids. Gels made of agarose, a natural seaweed product, are used to characterize nucleic acids that are 200 to 500,000 base pairs long.
Agarose gels, which can be purchased commercially, are prepared by dissolving purified agarose in warm electrophoresis buffer, cooling the solution to 50 °C (122 °F), and then pouring it into a mold, where it turns into a gel. Just as with polyacrylamide, the concentration of agarose in a gel determines the size of its pores. A comb placed in the gel before it sets produces the wells necessary for loading nucleic acid samples.
Nucleic acid fragments that are to be separated by size must be in "linearized" form. Plasmids, for example, must have their circular structure cut open using restriction enzymes before they are run on the gel. Otherwise, their rate of migration will depend on how supercoiled they are and whether they are nicked, instead of on their size. Nucleic acids that are separated in a gel can be seen with ethidium bromide or other stains.
Pulse-Field Gel Electrophoresis.
The conventional agarose gel electrophoresis described above separates nucleic acid fragments smaller than 50,000 base pairs (50 kilobase pairs). Pulse-field gel electrophoresis separates huge pieces of DNA that are between 200 and 3,000 kilobase pairs long. In this technique the electric field is not held constant during the separation. Instead, its direction and strength are repeatedly changed, with the molecules reorienting themselves every time the current changes. The molecules then slither like a snake through the gel matrix, in a process known as "reptation," with smaller fragments moving faster than larger ones. As the gel runs, it heats up and becomes more fluid, with the pulsing allowing the larger pieces to move more easily the longer the gel runs. Typically such gels are run overnight.
Once separated, large DNA pieces, such as complete genes, can be isolated for further experiments. They can be cloned into a bacterium, sequenced, or amplified by polymerase chain reaction.
Bloom, Mark V., Greg A. Freyer, and David A. Micklos. Laboratory DNA Science: An Introduction to Recombinant DNA Techniques and Methods of Genome Analysis. Menlo Park, CA: Addison-Wesley, 1996.
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