Gel Electrophoresis
DNA & protein electrophoresis
Chromatography – HPLC, GLC, TLC
PCR
AA Spectrometry
Gel Electrophoresis
Gel electrophoresis is the separation of deoxyribonucleic acid, ribonucleic acid, and protein through an electric charge. It is usually performed for analytical purposes, but may be used as a preparative technique to partially purify molecules prior to use of other methods such as mass spectrometry, PCR, cloning, DNA sequencing, or immuno-blotting for further characterization.
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.
Gel Electrophoresis Apparatus
DNA & protein electrophoresis
Electrophoresis
Protein electrophoresis is a sensitive analytical form of chromatography that allows the separation of charged molecules in a solution medium under the influence of an electric field. A wide range of molecules may be separated by electrophoresis, including, but not limited to DNA, RNA, and protein molecules.
The degree of separation and rate of molecular migration of mixtures of molecules depends upon the size and shape of the molecules, the respective molecular charges, the strength of the electric field, the type of medium used (e.g., cellulose acetate, starch gels, paper, agarose, polyacrylamide gel, etc.) and the conditions of the medium (e.g., electrolyte concentration, pH, ionic strength, viscosity, temperature, etc.).
Some mediums (also known as support matrices) are porous gels that can also act as a physical sieve for macromolecules.
In general, the medium is mixed with buffers needed to carry the electric charge applied to the system. The medium/buffer matrix is placed in a tray. Samples of molecules to be separated are loaded into wells at one end of the matrix. As electrical current is applied to the tray, the matrix takes on this charge and develops positively and negatively charged ends. As a result, molecules such as DNA and RNA that are negatively charged, are pulled toward the positive end of the gel.
Because molecules have differing shapes, sizes, and charges they are pulled through the matrix at different rates and this, in turn, causes a separation of the molecules. Generally, the smaller and more charged a molecule, the faster the molecule moves through the matrix.
When DNA is subjected to electrophoresis, the DNA is first broken by what are termed restriction enzymes that act to cut the DNA is selected places. After being subjected to restriction enzymes, DNA molecules appear as bands (composed of similar length DNA molecules) in the electrophoresis matrix. Because nucleic acids always carry a negative charge, separation of nucleic acids occurs strictly by molecular size.
Proteins have net charges determined by charged groups of amino acids from which they are constructed. Proteins can also be amphoteric compounds, meaning they can take on a negative or positive charge depending on the surrounding conditions. A protein in one solution might carry a positive charge in a particular medium and thus migrate toward the negative end of the matrix. In another solution, the same protein might carry a negative charge and migrate toward the positive end of the matrix. For each protein there is an isoelectric point related to a pH characteristic for that protein where the protein molecule has no net charge. Thus, by varying pH in the matrix, additional refinements in separation are possible.
The advent of electrophoresis revolutionized the methods of protein analysis. Swedish biochemist Arne Tiselius was awarded the 1948 Nobel Prize in chemistry for his pioneering research in electrophoretic analysis. Tiselius studied the separation of serum proteins in a tube (subsequently named a Tiselius tube) that contained a solution subjected to an electric field.
Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis techniques pioneered in the 1960s provided a powerful means of protein fractionation (separation). Because the protein bands did not always clearly separate (i.e., there was often a great deal of overlap in the protein bands) only small numbers of molecules could be separated. The subsequent development in the 1970s of a two-dimensional electrophoresis technique allowed greater numbers of molecules to be separated.
Two-dimensional electrophoresis is actually the fusion of two separate separation procedures. The first separation (dimension) is achieved by isoelectric focusing (IEF) that separates protein polypeptide chains according to amino acid composition. IEF is based on the fact that proteins will, when subjected to a pH gradient, move to their isoelectric point. The second separation is achieved via SDS slab gel electrophoresis that separates the molecule by molecular size. Instead of broad, overlapping bands, the result of this two-step process is the formation of a two-dimensional pattern of spots, each comprised of a unique protein or protein fragment. These spots are subsequently subjected to staining and further analysis.
Some techniques involve the application of radioactive labels to the proteins. Protein fragments subsequently obtained from radioactively labels proteins may be studied my radiographic measures.
There are many variations on gel electrophoresis with wide-ranging applications. These specialized techniques include Southern, Northern, and Western blotting. Blots are named according to the molecule under study. In Southern blots, DNA is cut with restriction enzymes then probed with radioactive DNA. In Northern blotting, RNA is probed with radioactive DNA or RNA. Western blots target proteins with radioactive or enzymatically tagged antibodies.
Modern electrophoresis techniques now allow the identification of homologous DNA sequences and have become an integral part of research into gene structure, gene expression, and the diagnosis of heritable and autoimmune diseases. Electrophoretic analysis also allows the identification of bacterial and viral strains and is finding increasing acceptance as a powerful forensic tool.
http://www.bookrags.com/Electrophoresis
Chromatography
Principles of Chromatography (General)
Chromatography is method of separating mixtures and identifying their components i.e. it's a separation method that exploits the differences in partitioning behavior of analytes between a mobile phase and a stationary phase to separate components in a mixture. Components of a mixture may be interacting with the stationary phase based on charge (ion-ion-interactions, ion-dipole-interactions), van der Waals' forces, relative solubility or adsorption (hydrophobic interactions, specific affinity). There are two theories of chromatography, the plate and rate theories
TLC (Thin Layer Chromatography)
Thin Layer Chromatography (TLC) is a widely-used chromatography technique used to separate chemical compounds [1]. It involves a stationary phase consisting of a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose immobilised onto a flat, inert carrier sheet. A liquid phase consisting of the solution to be separated dissolved in an appropriate solvent is drawn through the plate via capillary action, separating the experimental solution. It can be used to determine the pigments a plant contains, to detect pesticides or insecticides in food, in forensics to analyze the dye composition of fibers, or to identify compounds present in a given substance, among other uses. It is a quick, generic method for organic reaction monitoring.
http://en.wikipedia.org/wiki/Thin_layer_chromatography
HPLC (High Performance Liquid Chromatography)
High-performance liquid chromatography (HPLC) is a form of column chromatography used frequently in biochemistry and analytical chemistry. It is also sometimes referred to as high-pressure liquid chromatography. HPLC is used to separate components of a mixture by using a variety of chemical interactions between the substance being analyzed (analyte) and the chromatography column.
http://en.wikipedia.org/wiki/HPLC
GLC (Gas Liquid Chromatography)
Gas-liquid chromatography (GLC), or simply gas chromatography (GC), is a type of chromatography in which the mobile phase is a carrier gas, usually an inert gas such as helium or an unreactive gas such as nitrogen, and the stationary phase is a microscopic layer of liquid or polymer on an inert solid support, inside glass or metal tubing, called a column. The instrument used to perform gas chromatographic separations is called a gas chromatograph (also: aerograph, gas separator).
http://en.wikipedia.org/wiki/Gas_liquid_chromatography#Application
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