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. Immunoprecipitation Technique - Coursework Example

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Immunoprecipitation technique Immunoprecipitation technique Introduction Immunoprecipitation, as the name implies is a technique used for the precipitation i.e. purification or separation of certain proteins or antibodies from a mixture…
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Download file to see previous pages The basic idea behind this technique is the separation of one single protein from a mixture of proteins. This could give us the idea of several characters of a protein such as its relative occurrence in a solution, it’s up and down regulation as well as its affinity for a specific antibody. Technique/Methodology Usually the process of immunoprecipitation can be completed in two ways: in sequential or in one step. Commonly the antibody on which the required protein is supposed to be attached is immobilized on a solid support such as beads. This solution containing the immobilized antibody and the beaded support is then incubated with the solution that contains the protein mixture containing the required protein. Incubation allows the specific protein to bind to then antigen and form a complex. This complex can then be separated from the solution and studied with different techniques such as ELISA or Western Blot according to the requirements. (Pierce Biotechnology 2011) (Pierce Biotechnology 2011) The diagrams courtesy of ‘Pierce Biotechnology’ show the process of immunoprecipitation starting from preparation of a solid support along with the antigen to the incubation and the formation of the antigen-protein complexes till the precipitation of the required protein. Types There main types of immunoprecipitation are: 1. ...
pitation: Same as Chromatin only difference lies in the detection of RNA binding proteins Uses Immunoprecipitation has been useful in many aspects such as, It has enabled the scientists to know the activation of the proteins, their molecular weight and also separate some protein binding molecules too. This technique has also been helpful in detecting the abundance and activity of a protein. Protocols After collecting the required number of cells and washing them in ice cold PBS, the solution was spinned at 1000g at a temperature of 4 degrees. This would help in separating the supernatant fluid. The next step involves the resuspension of the pellet that contains the cells in an ice cold buffer after which the cells are lysed by centrifuge. The supernatant fluid is removed after spinning it again at 13000g at the same temperature for fifteen minutes. Bradford assay is then used to measure the quantity of the protein after the supernatant fluid is removed by spinning briefly. This supernatant free solution is then incubated in a cold room with the required amount of specific antibody solution. After the addition of Protein A or G beads to the tube it is again incubated for an hour and then spinned briefly again so that further supernatant is removed. This beaded fraction is then washed with ice cold buffer and spinned to remove the supernatant and the resultant solution is kept for further analysis. BCL-2 Proteins Bcl 2 family proteins have been identified to play a major role in the process of cell death i.e. apoptosis. These proteins play both anti and pro apoptotic roles. Some members of the family are supposed to increase while some are supposed to decrease this process of apoptosis. In this project the interaction of Bcl 2 family proteins with PUMA have been ...Download file to see next pagesRead More
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