Use of Sds-Page/Western Blot Techniques as Diagnostic Tools for Equine Medical Conditions

Topics: Western blot, Type I and type II errors, Molecular biology Pages: 9 (3736 words) Published: April 27, 2013
Use of SDS-PAGE/Western Blot Techniques as Diagnostic Tools for Equine Medical Conditions

SDS-PAGE Overview
Electrophoresis is commonly used to separate proteins using an applied electrical field. SDS-PAGE separates proteins by molecular weight, using sodium dodecyl sulfate (SDS; also known as lauryl sulfate) to denature them and a discontinuous polyacrylamide gel as a sieving matrix. The net charges of folded proteins are not dependent upon molecular weight. Rather, charge is determined by the sum of the individual amino acids’ R-group charges together with the molecular radius of the tertiary structure. Therefore, the tertiary structure must be destroyed and the effects of the charges offset. The SDS detergent disrupts the tertiary structure of proteins so that they become linear molecules. SDS carries a net negative charge within a wide pH range and binds with substantial uniformity to all the proteins’ positive charges, thereby dismantling the complex folded structures of the proteins. A reducing agent usually included in the SDS solution breaks any disulfide bonds. Finally, the proteins are in their linear forms and all carrying the same overall negative charge so that they will separate based on size. The polyacrylamide gel prevents the larger molecules from traveling as quickly as the smaller ones. It is chemically inert and can be mixed in various acrylamide concentrations to produce different pore sizes, thus offering a variety of separating conditions. SDS is also present in the gel to make sure that the proteins remain in their linear, negatively charged conformation. The gel is poured in two layers of differing pH: a near-neutral, low-acrylamide-content stacking gel ensures that all the proteins enter the running gel at the same time so that those of like molecular weight will travel as tight bands; the basic, higher-acrylamide running gel does the separating. The charge-to-mass ratio is nearly identical among the denatured proteins, so that although progress through the gel is caused by attraction to an anode (positively charged electrode), velocity is based almost entirely on molecular mass. The separated proteins may be visualized in the gel by staining, most often with Coomassie Brilliant Blue, an anionic dye that binds nonspecifically to proteins. It is usually mixed in an acidic solution that fixes the proteins in the gel as they are stained. After staining and destaining to remove excess dye, the proteins may be seen as blue bands on a clear or pale, translucent blue-purple background. SDS-PAGE can be used to estimate relative molecular mass of unknown proteins by comparison with known samples, as well as to approximate relative amounts of proteins in a mixed sample. The procedure may be employed to assess purity of fractions during a purification procedure. Moreover, separation by SDS-PAGE may be followed by such techniques as western blotting to separate further and identify proteins of interest in the sample, although the detection method used in the blotting procedure must be capable of recognizing denatured protein subunits.

Western Blot Overview
In Western blotting, also known as immunoblotting or protein blotting, selected proteins are transferred after SDS-PAGE from a polyacrylamide gel to a nitrocellulose or polyvinylidene fluoride membrane, creating an identical replica of the original pattern but enabling a wider variety of analytical procedures to be carried out. The technique enables rapid identification of target proteins at fairly low cost, using simple equipment and providing results that may be interpreted with relative ease. It has therefore become one of the most frequently used techniques in molecular biology, and is an important tool in both research and clinical settings. Transfer from gel to membrane is usually accomplished by electroblotting, although slower methods such as passive diffusion or vacuum-assisted transfer may also be used. In all types, a “sandwich” is made...
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