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Western Blots are typically done to determine the purity and MW of a protein. There are done to determine if a specific protein is in the sample. One does this by adding an antibody that is specific for the protein on interest. The antibody binds to the protein and can be detected various ways. Western blotting is effective and useful method to detect and characterize proteins in small amounts, such as clock proteins. Moreover, clock proteins’ other properties like half-life, molar amounts can also be found using western blotting. Advantages •Immunogenic responses from infectious agents (ex. viruses, bacteria) are hard to find since they are difficult to isolate from patient sample. But Western blotting can detect this. •Western Blotting utilizes not only antigens, but also antisera as a diagnostic tool. Antisera is widely used in the test for HIV presence. •Compared to ELISA, Western blotting has higher specificity; the higher specificity, the more the method is independent of the specificity of antibodies. •Polyvinylidene difluoride (PVDF), or Nylon, is often used as membrane in Western blotting, since it has a high protein-binding capacity and chemical stability. Even, some protein groups only bind to Nylon or favor strongly to it. •Among three common enzyme substrates, Fluorescent and Chemiluminescent create light detectable through X-ray or scanners. This ability enables high levels of sensitivity and quicker processing time. Disadvantages A non-intended protein has a slight chance of reacting with the secondary anti-body, resulting in the labeling of an incorrect protein. •Incidental phosphorylation or oxidation of proteins may result in multiple bands appearing after sample is processed. •The appearance of bubbles may occur when transferring the sample from the gel/membrane sandwich and may also occur when incubating the sample with antibodies, resulting in a skewed band reading. •If the transfer time is not sufficient when transferring proteins to the membrane, the larger proteins of higher molecular weight will not transfer properly, resulting in an incorrect or no band reading at all. •Too much methanol in the transfer buffer decreases the transfer efficiency of proteins from the gel to the membrane; however methanol aids in protein binding to several different membranes, so a correct balance is required. •Western Blotting is a very delicate process requiring the correct amounts of each component in order for successful identification of the presence of proteins. An imbalance in any step of the procedure may skew the entire process. ELISA,Enzyme-linked immunosorbent assay, is usually done to detect the presence of an antibody or an antigen in a sample. In ELISA an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal. The indirect ELISA utilizes an unlabeled primary antibody in conjunction with a labeled secondary antibody. Since the labeled secondary antibody is directed against all antibodies of a given species (e.g. anti-mouse), it can be used with a wide variety of primary antibodies (e.g. all mouse monoclonal antibodies). The use of secondary antibody also provides an additional step for signal amplification, increasing the overall sensitivity of the assay.