Gel Electrophoresisįor optimal separation, it is important to determine the ideal bisacrylamide:acrylamide ratio prior to electrophoresis. When the buffer contains absorbing materials, the Bradford assay (Bradford, 1976) can be used where a standard curve is created to determine unknown sample concentrations. Quantification for total protein can be achieved by measuring samples at 280 nm on a spectrophotometer, but the buffer must not contain absorbing materials. The protein concentrations of the samples to be loaded on a gel need to be determined. Importantly, quantification and comparison with other samples in western blot analysis is dependent on the protein lysates prepared for polyacrylamide gel electrophoresis. Lysis buffers used in sample preparation for western blotting should enable efficient protein extraction and maintain antisera recognition of the protein (MacPhee, 2010). Many of these inhibitors come in tablet form and can be simply dissolved in the lysis buffer before use. To avoid degradation of protein (proteases as well as phosphatases can be released during lysis), protease and phosphatase inhibitors can be included in the lysis buffer. There are many different lysis buffers, which can be chosen based on the protein of interest. Various detergents, salts, and buffers may be used to enable lysis of cells and to solubilize proteins. Tissue preparation can be performed at cold temperatures to avoid denaturation and degradation of protein. Solid tissue is mechanically broken down, usually using a homogenizer or by sonication in a lysis buffer (see below). It should also be noted that repeated freeze/thaw cycles can have an adverse effect on the quality of protein and should be avoided. Cells and tissues should be rapidly frozen with liquid nitrogen to avoid protease degradation of proteins or collected and lysed as quickly as possible. Protein can be measured from whole tissue or tissue culture extracts. The technique has continued to evolve, and there are many reports on troubleshooting and improving the technique (Kurien and Scofield, 2009). The proteins are then transferred to a membrane for detection using antibodies specific to the target protein. Basically, gel electrophoresis is used to separate native or denatured proteins. It is also called protein blotting or immunoblotting and has rapidly become a powerful tool for studying proteins. Burnette ( 1981) later employed the more widely used sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), which eventually led to this method being termed western blotting. ( 1979) enabled proteins to be electrophoretically separated using polyacrylamide–urea gels and transferred onto a nitrocellulose membrane. Western blotting evolved from Southern blotting, which is used to detect specific DNA sequences among DNA fragments separated by gel electrophoresis, and northern blotting, which is used to detect and quantify RNA and to determine its size, and also involves gel electrophoresis to separate RNA. The western (note that in this context “western” should be spelt with a lower-case “w”) blot is commonly used to identify, quantify, and determine the size of specific proteins.
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