Steps of the Western blot analysis Definition – define the term “Western blot analysis” Western blot analysis remains a technique that can be used for identifying certain proteins or antibodies in which proteins become separated by the use of electrophoresis, then become transferred to nitrocellulose, and finally reacted with antibody (Pound 2001, p. 38). 2) Principle of the method – briefly explain the principle of the Western blot: This must include the following; a) separation of protein b) The picking up of protein with antibody. Western blot often involves analysis of proteins from cells.
Often, cells become lysed to release protein content in them. These proteins content become separated in relation to their protein size by the use of a gel. Furthermore, the separated gel proteins become transferred onto a membrane surface by use of electricity. It becomes the purpose of the membrane to be used as a probe to separate proteins of interest. Often, a primary antibody becomes directed towards the protein of interest and binds to it leaving the nonspecific proteins unbound (Pound 2001, p. 39). Then, a secondary antibody becomes directed to recognize the primary antibody thereby forming a protein-antibody-antibody-sand witch.
Usually, the secondary antibody contain peroxidase enzyme that often converts luminal substrate to give a light releasing substrate. It hence becomes the light which becomes detected as a spot on the film. It becomes the spot size that determines how much protein remained in the spot, in relation to the size of other spots (Michael 2011, p. 56). 2) Steps of the Western blot analysis. List the steps and briefly describe/explain each of them. The first step in western blot analysis remains as the lysis of the cell.
Here, the cell becomes lysed with a lysic buffer which contains detergents responsible for breaking the cell’s membrane. In addition, the lysic buffer also contains an inhibitor of the photolytic enzymes which prevent enzymatic degradation of proteins (Rosenberg 2005, p. 66). The second step in western blot analysis involves temperature denaturation. In this step, a sample of protein becomes left to boil for five minutes at a temperature of 95 C. The purpose of that often became to allow protein denaturation to occur. The first step and second step sometimes become referred to as sample preparation steps since they involve preparation of the given proteins to be used in the third step (Rosenberg 2005, p. 66). The third step entails gel electrophoresis.
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