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

western Blot and its Applications - Essay ExampleWestern discolouration analysis passel detect your protein of interest from a mixture of a great payoff 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 program line such as untreated sample or a nonher cell type or tissue). It can analyze any protein sample whether from cells or tissues, but also can analyze recombinant proteins synthesized in vitro.Western imperfection is dependent on the quality of antibody you riding habit to probe for your protein of interest, and how specific it is for this protein.In western blotting, proteins atomic number 18 electrophoresed in polyacrylamide gel, transferred onto a nitrocellulose or nylon membrane, and the protein bands are detected by their specific interaction with antibodies, lectins or almost other compounds.First step involves the s eparation of tissue. Then proteins are separated by gel cataphoresis from sample. If you are passing play 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 hairlike 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 soci al occasion to be aware of is that proteins bind better to nitrocellulose at a low pH. You may expect to go through some trial-and-error to find the optimal pH. You also need to be current 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 employ probes for detecting specific antigens.2. Lectins are used as probes for the identification of glycoproteins.These probes may themselves be radioactive or a radioactive molecule may be tagged to them. Often the identification process is found on a Sandwich reaction. In such an approach, a species specific second antibody or protein A of staph 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 light tags, Steps in conducting a western blot.a single preparation of these labelled molecules can be employed as a general detector for various probes. Applications in different fieldsThe compliancy 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. distributively protein is labelled with a p followed by its molecular weight in thousands. 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 interrogation also uses Western blotting. The IgG Western Blot is a sandwich-type immunoassay performed in a style that allows visualization of the patients antibodies. It is a qualitative test and is generally more sensitive and specific than the ELISA. A despotic IgG result with clinical history may be indicative of