Western Blot and its Applications Essay Example | Topics and Well Written Essays - 1000 words

westward Blot and its Applications - Essay ExampleWestern blot analysis can detect your protein of interest from a intermixture of a great number of proteins. It can give you information about the size of your protein (with comparison to a size marker or ladder in kDa), and also give you information on protein expression (with comparison to a control such as untreated sample or another stall type or tissue). It can analyze any protein sample whether from cells or tissues, but also can analyze recombinant proteins synthesized in vitro.Western blot is dependent on the quality of antibody you use to probe for your protein of interest, and how particular it is for this protein.In western blotting, proteins be electrophoresed in polyacrylamide gel, transferred onto a nitrocellulose or nylon membrane, and the protein bands be detected by their specific interaction with antibodies, lectins or some other compounds.First step involves the separation of tissue. Then proteins be separate d by gel electrophoresis from sample. If you are acquittance to western blot for protein mass you can lyse in larger volumes If you are going to western blot a phospho-protein use phosphatase inhibitors. It will remove the phosphates from your proteins. If you are looking at protein-protein interactions use a less-stringent detergent such RIPA.By far the most common type of gel electrophoresis employs polyacrylamide gels and buffers loaded with sodium Dodecyl Sulphate (SDS). SDS PAGE allows separation of proteins by their molecular weight. Steps involved in obtaining protein for western blot.The protein bands are transferred onto a nitrocellulose or nylon membrane initially this was achieved by a capillary movement of buffer but now a days it is usually done by electrophoresis (electrophoretic blotting). The electrophoretic blotting, both of proteins and nucleic acids are much faster and more efficient than capillary blotting. One trendg to be aware of is that proteins bind better to nitrocellulose at a low pH. You may need to go through some trial-and-error to find the optimal pH. You also need to be sure there are no air bubbles between the nitrocellulose and the gel or your proteins will not transfer.The specific protein bands are identified in a variety of ways.1. Antibodies are the most commonly used probes for detecting specific antigens.2. Lectins are used as probes for the identification of glycoproteins.These probes may themselves be hot or a radioactive molecule may be tagged to them. Often the identification process is based on a Sandwich reaction. In such an approach, a species specific game antibody or protein A of Staphylococcus aureus or streptavidin is used to bind to the antibodies bound to the protein bands. These second molecules may be labelled with radioactive, enzyme or fluorescent tags, Steps in conducting a western blot.a single preparation of these labelled molecules can be employed as a general detector for various probes. Applica tions in different fieldsThe conformity HIV test uses a HIV western blot to detect anti- HIV. The HIV Western blot consists of a thin nitrocellulose strip in which are embedded proteins claimed to be unique to HIV. Each protein is labelled with a p followed by its molecular weight in thousands. blood serum is added to the strip and if there are antibodies to a particular protein this band will light up. The HIV Western blot is not standardised. Lyme disease testing also uses Western blotting. The IgG Western Blot is a sandwich-type immunoassay performed in a manner that allows visualization of the patients antibodies. It is a qualitative test and is generally more fine and specific than the ELISA. A positive IgG result with clinical history may be indicative of