Gel electrophoresis
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| Gel electrophoresis | |
 Gel electrophoresis apparatus - An agarose gel is placed in this buffer-filled box and electrical current is applied via the power supply to the rear. The negative terminal is at the far end (black wire), so DNA migrates toward the camera. |
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| Classification | Electrophoresis |
|---|---|
| Other Techniques | |
| Related | Capillary electrophoresis SDS-PAGE Two-dimensional gel electrophoresis Temperature gradient gel electrophoresis |
Gel electrophoresis is a technique used for the separation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein molecules using an electric current applied to a gel matrix.[1] It is usually performed for analytical purposes, but may be used as a preparative technique prior to use of other methods such as mass spectrometry, RFLP, PCR, cloning, DNA sequencing, or Southern blotting for further characterization.
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[edit] Separation
The term "gel" in this instance refers to the matrix used to contain, then separate the target molecules. In most cases the gel is a crosslinked polymer whose composition and porosity is chosen based on the specific weight and composition of the target to be analyzed. When separating proteins or small nucleic acids (DNA, RNA, or oligonucleotides) the gel is usually composed of different concentrations of acrylamide and a cross-linker, producing different sized mesh networks of polyacrylamide. When separating larger nucleic acids (greater than a few hundred bases), the preferred matrix is purified agarose. In both cases, the gel forms a solid, yet porous matrix. Acrylamide, in contrast to polyacrylamide, is a neurotoxin and must be handled using appropriate safety precautions to avoid poisoning. Another gel matrix is agarose, long unbranched chains of uncharged carbohydrate without cross links giving a gel with large pores allowing separation of macromolecules and macromolecular complexes.
"Electrophoresis" refers to the electromotive force (EMF) that is used to move the molecules through the gel matrix. By placing the molecules in wells in the gel and applying an electric current, the molecules will move through the matrix at different rates, determined largely by their mass when the charge to mass ratio (Z) of all species is uniform, toward the anode if negatively charged or toward the cathode if positively charged [2].
[edit] Visualization
After the electrophoresis is complete, the molecules in the gel can be stained to make them visible. Ethidium bromide, silver, or coomassie blue dye may be used for this process. Other methods may also be used to visualize the separation of the mixture's components on the gel. If the analyte molecules fluoresce under ultraviolet light, a photograph can be taken of the gel under ultraviolet lighting conditions. If the molecules to be separated contain radioactivity added for visibility, an autoradiogram can be recorded of the gel.
If several mixtures have initially been injected next to each other, they will run parallel in individual lanes. Depending on the number of different molecules, each lane shows separation of the components from the original mixture as one or more distinct bands, one band per component. Incomplete separation of the components can lead to overlapping bands, or to indistinguishable smears representing multiple unresolved components.
Bands in different lanes that end up at the same distance from the top contain molecules that passed through the gel with the same speed, which usually means they are approximately the same size. There are molecular weight size markers available that contain a mixture of molecules of known sizes. If such a marker was run on one lane in the gel parallel to the unknown samples, the bands observed can be compared to those of the unknown in order to determine their size. The distance a band travels is approximately inversely proportional to the logarithm of the size of the molecule.
[edit] Applications
Gel electrophoresis is used in forensics, molecular biology, genetics, microbiology and biochemistry. The results can be analyzed quantitatively by visualizing the gel with UV light and a gel imaging device. The image is recorded with a computer operated camera, and the intensity of the band or spot of interest is measured and compared against standard or markers loaded on the same gel. The measurement and analysis are mostly done with specialized software.
Depending on the type of analysis being performed, other techniques are often implemented in conjunction with the results of gel electrophoresis, providing a wide range of field-specific applications.
[edit] Nucleic acids
In the case of nucleic acids, the direction of migration, from negative to positive electrodes, is due to the naturally-occurring negative charge carried by their sugar-phosphate backbone.[3]
Double-stranded DNA fragments naturally behave as long rods, so their migration through the gel is relative to their radius of gyration, or, for non-cyclic fragments, size. Single-stranded DNA or RNA tend to fold up into molecules with complex shapes and migrate through the gel in a complicated manner based on their tertiary structure. Therefore, agents that disrupt the hydrogen bonds, such as sodium hydroxide or formamide, are used to denature the nucleic acids and cause them to behave as long rods again.[4]
Gel electrophoresis of large DNA or RNA is usually done by agarose gel electrophoresis. See the "Chain termination method" page for an example of a polyacrylamide DNA sequencing gel. Characterization through ligand interaction of nucleic acids or fragments may be performed by mobility shift affinity electrophoresis.
[edit] Proteins
Proteins, unlike nucleic acids, can have varying charges and complex shapes, therefore they may not migrate into the polyacryl amide gel at similar rates, or at all, when placing a negative to positive EMF on the sample. Proteins therefore, are usually denatured in the presence of a detergent such as sodium dodecyl sulfate/sodium dodecyl phosphate (SDS/SDP) that coats the proteins with a negative charge.[1] Generally, the amount of SDS bound is relative to the size of the protein (usually 1.4g SDS per gram of protein), so that the resulting denatured proteins have an overall negative charge, and all the proteins have a similar charge to mass ratio. Since denatured proteins act like long rods instead of having a complex tertiary shape, the rate at which the resulting SDS coated proteins migrate in the gel is relative only to its size and not its charge or shape.[1]
Proteins are usually analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), by native gel electrophoresis, by quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE), or by 2-D electrophoresis.
Characterization through ligand interaction may be performed by electroblotting or by affinity electrophoresis in agarose or by capillary electrophoresis as for estimation of binding constants and determination of structural features like glycan content through lectin binding.
[edit] History
- 1930s - first reports of the use of sucrose for gel electrophoresis
- 1955 - introduction of starch gels, mediocre separation
- 1959 - introduction of acrylamide gels (Raymond and Weintraub); accurate control of parameters such as pore size and stability
- 1964 - disc gel electrophoresis (Ornstein and Davis)
- 1969 - introduction of denaturing agents especially SDS separation of protein subunit (Weber and Osborn)[5]
- 1970 - Laemmli separated 28 components of T4 phage using a stacking gel and SDS
- 1975 - 2-dimensional gels (O’Farrell); isoelectric focusing then SDS gel electrophoresis
- 1977 - sequencing gels
- late 1970s - agarose gels
- 1983 - pulsed field gel electrophoresis enables separation of large DNA molecules
- 1983 - introduction of capillary electrophoresis
A 1959 book on electrophoresis by Milan Bier cites references from the 1800s.[6] However, Oliver Smithies made significant contributions. Bier states: "The method of Smithies ... is finding wide application because of its unique separatory power." Taken in context, Bier clearly implies that Smithies' method is an improvement.
[edit] See also
- Affinity electrophoresis
- DNA electrophoresis
- Electroblotting
- Electrofocusing
- Gel isolation
- Native Gel Electrophoresis
- Northern blotting
- Protein electrophoresis
- SDS PAGE
- Southern blotting
- Two-dimensional gel electrophoresis
- Western blotting
- Eastern blotting
- Zymography
[edit] References
- ^ a b c Berg JM, Tymoczko JL Stryer L (2002). Molecular Cell Biology (5th ed. ed.). WH Freeman. ISBN 0-7167-4955-6. http://www.ncbi.nlm.nih.gov/books/bv.fcgi?&rid=stryer.section.438#455.
- ^ Robyt, John F.; White, Bernard J. (1990). Biochemical Techniques Theory and Practice. Illinois: Waveland Press. ISBN 0-88133-556-8.
- ^ Lodish H, Berk A, Matsudaira P, et al (2004). Molecular Cell Biology (5th ed. ed.). WH Freeman: New York, NY. ISBN 978-0716743668. http://www.ncbi.nlm.nih.gov/books/bv.fcgi?&rid=mcb.section.1637#1648.
- ^ Troubleshooting DNA agarose gel electrophoresis. Focus 19:3 p.66 (1997).
- ^ http://www.ncbi.nlm.nih.gov/pubmed/5806584
- ^ Milan Bier (ed.) (1959). Electrophoresis. Theory, Methods and Applications (3rd printing ed.). Academic Press. pp. 225. LCC 59-7676. OCLC 1175404.
[edit] External links
- Biotechniques Laboratory electrophoresis demonstration, from the University of Utah's Genetic Science Learning Center
- Discontinuous native protein gel electrophoresis
- Drinking straw electrophoresis
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