Electrophoresis: Difference between revisions
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== Detection Techniques == | == Detection Techniques == | ||
There are several detection techniques used to visualize bands. The most commonly used technique involves chemically staining proteins with coomassie blue or silver. Another method covalently bonds proteins to fluorescent molecules before electrophoresis. | |||
Nucleic acids are commonly visualized by illuminating the bands under a UV light after binding them to ethidium bromide <ref name=RickwoodP />. Other methods include: chemical staining, fluorescence, radioactivity, immunoelectrophoretic techniques and on-column/end-column detection <ref name=Harrison />. | |||
Staining is an easy way to make a rough estimate of the quantity, purity and molecular weight of a compound. In general, the intensity of the staining is proportional to the concentration of the compound <ref name=Chen>Chen, K., Glase, J. (2009-2010). Chapter 13 – DNA Technology: From Recombination to Genomic Sequencing and Forensic Analysis. In Chen, K. & Hester, L. L. (Eds.), Investigative Biology a laboratory text (pp. 245-288). Plymouth, MI: Hayden McNeil.</ref>. Generally, darker bands contain a larger quantity. | |||
== Gel Electrophoresis of DNA and RNA == | == Gel Electrophoresis of DNA and RNA == | ||
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Electrophoresis is a separation technique frequently used in the analysis of proteins and nucleic acids. The process known as electrophoresis, involves the migration of particles or molecules (in particular proteins, DNA, and RNA) through an electric field that separates them exclusively on the basis of their size or molecular weight. The direction the molecule moves depends on its charge while the rate of migration is affected by the size, shape, density of the gel and the strength of the applied current (5c).
Electrophoresis is a very simple process and relatively quick with a high resolution. In addition electrophoresis is an extremely useful method to estimate the purity of a sample. The technique is also very sensitive to slight variations in molecular weight, size, and even shape of nucleic acids and proteins [1]. Electrophoresis can also be useful when it doesn’t affect the molecule’s structure or denature the protein Cite error: Invalid <ref> tag; invalid names, e.g. too many
The Process
As shown in the diagram, the nucleic acids or proteins are loaded into the wells or depressions at one end on the eletrophoretic medium (also known as a ‘’gel’’). The apparatus also has two electrodes on either side of the eletrophoretic medium. The anode is positively charged while the cathode is negatively charged. When a power source connects the two electrodes the charged particles begin to migrate towards the oppositely charged electrode due to the electric potential field within the media [1].
The velocity of the particles are related to the electric field potential by the following equation:
- Failed to parse (SVG (MathML can be enabled via browser plugin): Invalid response ("Math extension cannot connect to Restbase.") from server "https://wikimedia.org/api/rest_v1/":): {\displaystyle uμ = \frac{v}{E}\ }
Where E is the electric field potential that provides the driving force on the particle. μ is the electrophoretic mobility and v is the velocity of the particle [1]. For proteins, the equation can also be written as
- Failed to parse (syntax error): {\displaystyle uμ = \frac{v}{E}\ = \frac{Z}{f}\ }
Where Z is the protein’s net charge and f is a frictional coefficient related to the protein’s shape [2].
Smaller molecules move faster in the gel than larger molecules and therefore they end up closer to the positive anode. Molecules that are about the same size move at the same rate through the electrophoretic medium. The figure to the right shows the molecules in ‘’bands’’. The column on the far left contains bands with known molecular weights. This is useful to determine the molecular weight of an unknown particle [2].
Generation of Heat in Electrophoresis Instrumentation
Due to the electric field in electrophoresis, the equipment generates a large amount of heat that needs to be dissipated for maximum efficiency. Since the gel’s viscosity and density changes with an increasing temperature, it is important to remove as much heat as possible from the apparatus otherwise the gel will melt. As a solution, increasing the surface area to volume ratio of the gel usually helps to dissipate the heat. For instance, capillary electrophoresis efficiently removes heat because of its high surface-area to volume ratio. Similar to native electrophoresis, this commonly used method maintains a constant electric field at a stable pH where the separation depends upon mobility [1].
Electrophoretic Mediums
One of the major factors affecting the separation of nucleic acids or proteins is the density of the gel. Polyacrylamide Gels are the most commonly used gels for electrophoresis and are mainly used to separate smaller proteins or fragments of nucleic acids (with molecular weights as low as 2000). Agarose gels on the other hand are used to separate much larger molecules (above a molecular weight of 106) [3]. As the percentage of agarose or polyacrylamide increases in the solution, the gel becomes denser. As a result, the nucleic acids or proteins move slower since the pore size has decreased. The resolution or distance between the fragments (or bands) relative to their width, therefore, depends on the density of the gel [3]. The density of the gel chosen is usually one that adequately separates all the components of interest.
Detection Techniques
There are several detection techniques used to visualize bands. The most commonly used technique involves chemically staining proteins with coomassie blue or silver. Another method covalently bonds proteins to fluorescent molecules before electrophoresis.
Nucleic acids are commonly visualized by illuminating the bands under a UV light after binding them to ethidium bromide [3]. Other methods include: chemical staining, fluorescence, radioactivity, immunoelectrophoretic techniques and on-column/end-column detection [1].
Staining is an easy way to make a rough estimate of the quantity, purity and molecular weight of a compound. In general, the intensity of the staining is proportional to the concentration of the compound [4]. Generally, darker bands contain a larger quantity.
Gel Electrophoresis of DNA and RNA
Gel Electrophoresis of Proteins
Native Electrophoresis
Denaturing Gel electrophoresis
Disadvantage/Advantage
Isoelectric focusing
Two-Dimensional Electrophoresis for Proteins
Two-Dimensional Electrophoresis for DNA and RNA
Blotting Techniques
Advantages & Disadvantages of Electrophoresis
References
- ↑ 1.0 1.1 1.2 1.3 1.4 Harrison, R. G., Todd, P., Rudge S. R., Petrides, D. P. (2003). Bioseparations Science and Engineering. New York, NY: Oxford University Press.
- ↑ 2.0 2.1 Nelson, D. L., Cox, M. M. (2008). Lehninger Principles of Biochemistry (5th ed.). New York, NY: W.H. Freeman and Company.
- ↑ 3.0 3.1 3.2 Rickwood, D. & B.D. Hames (Eds.). (1982). Gel electrophoresis of proteins: a practical approach. Oxford: IRL Press Limited.
- ↑ Chen, K., Glase, J. (2009-2010). Chapter 13 – DNA Technology: From Recombination to Genomic Sequencing and Forensic Analysis. In Chen, K. & Hester, L. L. (Eds.), Investigative Biology a laboratory text (pp. 245-288). Plymouth, MI: Hayden McNeil.