Steps of the Western Blot Analysis - Essay Example

? 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. In order to separate the different proteins from each other, the lysades became loaded on SDS polyacrylamide gel. SDS as a component of the gel allowed for neutralization of the proteins thereby making their separations entirely based on molecular weight (Kontermann 2002, p.34). The separation became performed by use of constant voltage. In order to visualize, the use of a dye called bromophenol blue became used (Rosenberg 2005, p.66). The fourth step in western blot analysis involved blotting. In here, blotting refers to the direct transfer of the given proteins to the membrane from the gel by use of electric power plus nitrocellulose. The membrane pores of nitrocellulose permanently accommodate the given proteins transfer from the gel (Rosenberg 2005, p.66). More over, the fifth step in western blotting analysis involves blocking. This process involves filling with empty pores of the given nitrocellulose membrane with some certain proteins that will not be recognized by antibodies used commonly in the experiment (BSCL bovine serum albumin) (Rosenberg 2005, p.66). The sixth step entails incubation with primary antibody. In here, blocked membranes become incubated in a solution containing primary antibody that got the capability of recognizing target proteins. Normally, the primary antibody often becomes dissolved in the given buffer used for deblocking. The primary antibody often permanently binds to the given target protein (Rosenberg 2005, p.67). The seventh step in western blot analysis entails application of the secondary antibody. Here, a secondary antibody becomes applied after primary antibody incubation. The secondary antibody often goes and binds to the primary antibody forming a protein-antibody-antibody sand witch. Normally, the secondary antibody becomes labeled with florescent dye for the purpose of visibility (Rosenberg 2005, p.67). Therefore, the eighth procedure in western blot analysis involves visualization. Here, the visualization of the target protein and antibodies become possible. This could be achieved by the help of fluorescent scanning. 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 (Rosenberg 2005, p.67). In the ninth step, in western blot analysis, involves detection of quantity of protein found in a given spot. Here, they detect a protein constant in all types of proteins, and that is actin. It becomes the spot size that determines how much protein remained in the spot, in relation to the size of other spots. 3) Explain the necessity of the loading control (important) The commonly used loading control in western blot analysis remains as actins. In order for one to interpret the given western blot analysis results; a loading control always serves a vital purpose throughout the experiment. A loading control always ensures that the same amount of the given protein sample used ends loaded in each lane. Furthermore, the loading control ensures proteins get transferred from the given gel to the membrane while keeping equal efficiency among the different lanes. Moreover, the loading control ensures primary antibody and secondary antibody undergo the process of incubation very well. In addition, it also ensures signal detection becomes uniform across the different lanes. 4) Comment on the specificity of the Western blot. Western blot analysis remains to be an excellent experiment since antibodies often become directed towards certain specific proteins. Specificity of the experiment remains quit high as a primary and secondary antibody become used in the experiment (King 2003, p.16). The first antibody ensures it binds to the rightful protein while the secondary antibody recognizes where the first went and colors it. Western blot analysis often recommended in diagnosis of conditions or diseases that require high specifity through the formation of antibody antigen reaction. Bibliography King, D., 2003, Applications and engineering of monoclonal antibodies, New York: Taylor & Francis. Kontermann, R., 2002, Antibody Engineering, New York: Springer. Michael Stumpf, D. J., 2011, Handbook of Statistical Systems Biology, New York: John Wiley and Sons. Pound, J., 2001, Immunochemical Protocols, Massachussets: Springer. Rosenberg, I. M., 2005, Protein analysis and purification: benchtop techniques, New York: Springer. Read More