Electrophoresis Assignment Help
Separation of proteins based on the migration of charged proteins in an electric field. It is not used to purify proteins in large amounts as it often adversely affects the structure and thus the function of proteins. Its advantage is that protein can be visualized as well as separates and permitting a researcher to finding quickly the number of different proteins in a mixture or the degree of purity of a protein preparation. It also allows the determination of iso-electric point, molecular weight of protein. The migration of a protein in a gel during electrophoresis is a function of its size and shape. The method makes the use of a detergent Sodium dodecyl sulphate (SDS) proportional to the molecular weight of protein. SDS binding unfolds the protein such that the bound proteins assume a similar shape. After electrophoresis, the proteins are visualized by adding dyes such as Coomassie blue which binds to the protein and not to the gel.
Agrose gel electrophoresis is a method used in biochemistry and molecular biology to separating DNA and RNA molecules by their size . This is achieved by moving negatively charged nucleic acid molecules through an agrose matrix with an electric field which is also called electrophoresis . Smaller molecules move faster and migrate farther than longer ones. The technique is used for estimation of the size of DNA molecules following restriction enzymolysis , such as in restriction mapping of DNA cloning , PCR products analysis , such as in molecular genetic diagnosis and genetic fingerprinting , restricted genome DNA separation from prior to Southern transfer and of RNA prior to Northern transfer. The most common dye agrose gel is used to make visible DNA or RNA bands for electrophoresis is ethidium bromide, usually abbreviated as EtBr. In UV light , Its fluoresces ,is intercalated into DNA or RNA . In UV-light , DNA is running through an EtBr-treated gel and visualizing , any band containing more than and equal to 20ng DNA becomes distinctly visible.
The various techniques through which it is achieved are discussed in brief as follows:
- Mass Spectrometry
- Isoelectric Focussing
- Scatchard Analysis
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