Coursework. Immunoprecipitation Technique Coursework

. Immunoprecipitation Technique - Coursework typesetters caseThe basic idea behind this technique is the separation of one single protein from a mixture of proteins. This could relent us the idea of several characters of a protein such as its relative occurrence in a solution, its up and down regulation as well as its affinity for a special(prenominal) antibody. Technique/Methodology Usually the demonstrate of immunoprecipitation can be completed in two shipway in sequential or in one step. Comm besides the antibody on which the required protein is supposed to be attached is immobilized on a solid turn out 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 biotech 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 establishment of the antigen-protein complexes till the precipitation of the required protein. Types There main types of immunoprecipitation be 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 cope the activation of the proteins, their molecular weight and also separate some protein binding molecules too. This technique has also been right-hand in detecting the abundance and activity of a protein. Protocols After collecting the required number of cells and airstream them in ice cold PBS, the solution was spinned at 1000g at a temperature of 4 degrees. This woul d 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 withdraw after spinning it again at 13000g at the akin 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 get on with the required amount of specific antibody solution. After the addition of Protein A or G beads to the resistance it is again incubated for an hour and then spinned briefly again so that further supernatant is removed. This beaded portion is then washed with ice cold buffer and spinned to remove the supernatant and the resultant solution is unplowed 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. apopt osis. These proteins play both anti and pro apoptotic roles. Some members of the family are supposed to improver while some are supposed to decrease this process of apoptosis. In this project the interaction of Bcl 2 family proteins with PUMA have been