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