Molecular Biology

Separation Science Dna Rna Protein



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Page (Polyacrylamide Gel Electrophoresis) and Western Blots

Gel Electrophoresis is a separation science commonly used in many laboratories when searching for protein or DNA of interest. It is similar to using strainers in your kitchen to separate large pieces of food from the smaller pieces. Gel electrophoresis performs the same task but with the commonly used laboratory material acrylamide as the strainer. Acrylimde is a polymer which can form a network of cross- linking fibers when used in conjunction with a cross-linking agent. Together a three dimensional lattice called a polyacrylamide gel is formed. Protein molecules can migrate through this lattice by the applied force of an electric field, much the same way grain moves through a strainer by the force gravity.

When a strainer is not able to separate the food of interest from what is not needed, one reaches for a finer size strainer. The same is obtained when adjusting the amount or percentage of acrylamide in the gel. Adjusting the porosity of the gel aids the laboratory worker in isolating proteins or molecules of a particular size. Using a higher order of cross-linking monomers will result in smaller pores while using less results in larger pores. A protein's Stoke's radius (molecular weight range) can help identify or isolate a protein in accordance to the features of the gel's porosity.

One can create gels with more needed features. In general proteins traverse polyacrylamide gels according to charge and size. However this criteria may not be enough or too much in isolating a protein of interest. Many may want to simplify the search according to size alone and this can be accomplished by adding sodium dodecyl sulfate (SDS) to the gel. SDS, commonly known as a detergent, coats the molecule with its endogenous charge characteristic making all protein molecules in the unknown fraction sharing the same charge. Thus the separation performed during the gel electrophoresis is done strictly by a protein's mass.

Proteins resolved in a gel by electrophoresis are going to be invisible to the naked eye. Therefore dye additives like Coomassice Brilliant Blue are used to make the bands of protein traversing the gel visible for quantitative and qualitative purposes along with a marker. A marker is much like a legend where known proteins of particular molecular weight and concentration are run along with the unknown protein to determine size and concentration of the unknown.

Western Blotting is another modification to polyacrylamide gels where it is possible to isolate proteins that interact with particular antibodies. Western blotting utilizes SDS-Page technique and combines it in a gel sandwich with a membrane containing the antibody of interest. The protein on the SDS treated gel will migrate out of the gel and onto the antibody treated membrane if protein-antibody interaction in attained. A series of antibodies can be added for further isolation of proteins according to particular properties and interactions.

Gel electrophoresis is an excellent way to isolate biological molecules such as DNA, protein, or RNA and can be done through the basic PAGE method or more advanced methods of electrophoresis like Western blotting, Isoelectric Focusing, or Two-dimensional gel electrophoresis. Electing other disciplines such as spectroscopy, chromatography, and cloning can either help in positive identification of the protein or provide multiple copies of the protein for further experimentation.

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