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Gel electrophoresis
About: Gel electrophoresis is a research topic. Over the lifetime, 26026 publications have been published within this topic receiving 1113565 citations.
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TL;DR: The isolated oxygenase catalyzed the insertion of both oxygen molecules required for the formation of prostaglandins and thromboxanes from polyunsaturated fatty acid substrates in a specific activity of 46,000 units/mg of protein.
416 citations
TL;DR: Medium conditioned by human placental tissue was found to stimulate granulocytic and monocytic colony formation by human marrow cells in semisolid agar cultures and the active factor was not dependent on the presence of adherent marrow cells with endogenous colony-stimulating activity.
415 citations
TL;DR: The isolate of prostaglandin endoperoxide synthetase requires haemin, which possibly acts as an easily dissociable prosthetic group, and a suitable hydrogen donor to protect the enzyme from peroxide inactivation and which is consumed in stoichiometric amounts to reduce the intermediate hydroperoxy group.
414 citations
TL;DR: Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb and students should be able to understand the mechanism by which DNA fragments are separated within a gel matrix.
Abstract: Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel's molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to:
1. Understand the mechanism by which DNA fragments are separated within a gel matrix
2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix
3. Identify an agarose solution of appropriate concentration for their needs
4. Prepare an agarose gel for electrophoresis of DNA samples
5. Set up the gel electrophoresis apparatus and power supply
6. Select an appropriate voltage for the separation of DNA fragments
7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands
8. Determine the sizes of separated DNA fragments
413 citations
TL;DR: Electrophoretic transfer (electroblotting) of proteins or their cleavage fragments onto activated glass filter paper sheets immediately after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis has been successfully applied to the sequencing of a variety of proteins and peptides isolated from one-dimensional and two-dimensional polyacylamide gels.
410 citations