Gel electrophoresis



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.
Classification Electrophoresis
Other Techniques
Related Two-dimensional gel electrophoresis
Temperature gradient gel electrophoresis

Gel electrophoresis is a technique used for the separation of Southern blotting for further characterization.

Separation

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"anode if negatively charged [2].

Visualization

  After the electrophoresis is complete, the molecules in the gel can be stained to make them visible. 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.

Applications

Gel electrophoresis is used in 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.

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 formamide, are used to denature the nucleic acids and cause them to behave as long rods again.[4]

Gel electrophoresis of large polyacrylamide DNA sequencing gel.

Proteins

  Proteins, unlike nucleic acids, can have varying charges and complex shapes, therefore they may not migrate into the gel at similar rates, or at all, when placing a negative to positive EMF on the sample. Proteins therefore, are usually 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]

2-D electrophoresis.

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 protein subunit (Beber and Osborn)
  • 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 moleucles
  • 1983 - introduction of capillary electrophoresis

A 1959 book on electrophoresis by Milan Bier cites references from the 1800s.[5] 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.

See also

References

  1. ^ a b c Berg JM, Tymoczko JL Stryer L (2002). Molecular Cell Biology, 5th ed., WH Freeman. ISBN 0-7167-4955-6. 
  2. ^ Robyt, John F. (1990). Biochemical Techniques Theory and Practice. Waveland Press. ISBN 0-88133-556-8. 
  3. ^ Lodish H, Berk A, Matsudaira P, et al (2004). Molecular Cell Biology, 5th ed., WH Freeman: New York, NY. ISBN 978-0716743668. 
  4. ^ Troubleshooting DNA agarose gel electrophoresis. Focus 19:3 p.66 (1997).
  5. ^ Milan Bier (ed.) (1959). Electrophoresis. Theory, Methods and Applications, 3rd printing, Academic Press, 225. LCC 59-7676. OCLC 1175404. 


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This article is licensed under the GNU Free Documentation License. It uses material from the Wikipedia article "Gel_electrophoresis". A list of authors is available in Wikipedia.